Enzymes useful for peracid production

ABSTRACT

Acetyl xylan esterases and variants thereof having perhydrolytic activity are provided for producing peroxycarboxylic acids from carboxylic acid esters and a source of peroxygen. Multi-component peracid generation systems comprising an enzyme catalyst having perhydrolytic activity are also provided, as are methods of using the present enzyme catalyst to produce peroxycarboxylic acids. The polypeptide having perhydrolytic activity may be used to produce peroxycarboxylic acids suitable for use in a variety of applications such as cleaning, disinfecting, sanitizing, bleaching, wood pulp processing, paper pulp processing, and personal care applications.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/618,397 filed Mar. 30, 2012, which is incorporated by reference inits entirety.

TECHNICAL FIELD

This disclosure relates to the field of peroxycarboxylic acidbiosynthesis and enzyme catalysis. More specifically, multi-componentperacid generation systems comprising an enzyme catalyst havingperhydrolytic activity are provided. Methods of using the present enzymecatalyst to produce peroxycarboxylic acids are also provided.

BACKGROUND

Peroxycarboxylic acid compositions can be effective antimicrobialagents. Methods of using peroxycarboxylic acids to clean, disinfect,and/or sanitize hard surfaces, textiles, meat products, living planttissues, and medical devices against undesirable microbial growth havebeen described (U.S. Pat. No. 6,545,047; U.S. Pat. No. 6,183,807; U.S.Pat. No. 6,518,307; U.S. Patent Application Publication No.2003-0026846; and U.S. Pat. No. 5,683,724). Peroxycarboxylic acids havealso been used in various bleaching applications including, but notlimited to, wood pulp bleaching/delignification and laundry careapplications (European Patent 1040222B1; U.S. Pat. No. 5,552,018; U.S.Pat. No. 3,974,082; U.S. Pat. No. 5,296,161; and U.S. Pat. No.5,364,554). The desired efficacious concentration of peroxycarboxylicacid may vary according to the product application (for example, ca. 500ppm to 1000 ppm for medical instrument disinfection, ca. 30 ppm to 80ppm for laundry bleaching or disinfection applications) in 1 min to 5min reaction time at neutral pH.

Enzymes structurally classified as members of family 7 of thecarbohydrate esterases (CE-7) have been employed as perhydrolases tocatalyze the reaction of hydrogen peroxide (or alternative peroxidereagent) with alkyl esters of carboxylic acids in water at a basic toacidic pH range (from ca. pH 10 to ca. pH 5) to produce an efficaciousconcentration of a peroxycarboxylic acid for such applications asdisinfection (such as medical instruments, hard surfaces, textiles),bleaching (such as wood pulp or paper pulp processing/delignification,textile bleaching and laundry care applications), and other laundry careapplications such as destaining, deodorizing, and sanitization, andpersonal care applications (U.S. Pat. Nos. 7,964,378; 7,951,566; and7,723,083; Published U.S. Patent Application No. 2008-0176299 toDiCosimo et al.; and Published U.S. Patent Application Nos. 2012-0317733and 2012-0328534 to Chisholm et al.). The CE-7 enzymes have been foundto have high specific activity for perhydrolysis of esters, particularlyacetyl esters of alcohols, diols and glycerols. Variant CE-7perhydrolases derived from several species having improved performancebeen reported by DiCosimo et al. (U.S. Pat. Nos. 7,927,854; 7,923,233;7,932,072; 7,910,347; 7,960,528; 8,062,875; 8,206,964; 8,389,254; and8,389,255; and Published U.S. Patent Application Nos. 2011-0236336 and2011-0236338).

Previously reported CE-7 carbohydrate esterases having perhydrolyticactivity (both wild type and variants thereof) comprised a conservedstructural “signature” motif as defined by Vincent et al. (J. Mol.Biol., 330:593-606 (2003)).

More specifically, the CE-7 signature motif used to structurallyidentify and define members of the CE-7 carbohydrate esterase familycomprises three conserved submotifs: 1) an “RGQ” submotif ofArg118-Gly119-Gln120, 2) a “GXSQG” submotif ofGly186-Xaa187-Ser188-Gln189-Gly190, and 3) an “HE” submotif ofHis303-Glu304 (residue numbering and orientation relative to theThermotoga maritima reference sequence provided as SEQ ID NO: 2).

While the vast majority of enzymes classified as CE-7 carbohydrateesterases are comprised of the signature motif defined by Vincent etal., several polypeptide sequences have been added to family 7 of thecarbohydrate esterases that do not contain the “HE” submotif (Cantarelet al., “The Carbohydrate-Active EnZymes database (CAZy): an expertresource for Glycogenomics”, NAR, 37:D233-D238 (2009)). The presence ofperhydrolytic activity within this subgroup has not been reported.

Incorporation of perhydrolytic enzyme technology into some applicationsmay require the identification of new perhydrolytic enzymes. As such,there remains a need to identify additional enzyme catalysts comprisingpolypeptide having significant perhydrolytic activity.

SUMMARY

Several enzymes have been identified having perhydrolytic activitysuitable for the production of peracids at efficacious concentrations.

In one embodiment, an enzymatic peracid generation system is providedcomprising a set of reaction components comprising:

-   -   (1) at least one substrate selected from the group consisting        of:        -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   wherein            -   X=an ester group of the formula R₆—C(O)O;            -   R₆=a C1 to C7 linear, branched or cyclic hydrocarbyl                moiety, optionally substituted with hydroxyl groups or                C1 to C4 alkoxy groups, wherein R₆ optionally comprises                one or more ether linkages for R₆═C2 to C7;            -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl                moiety or a five-membered cyclic heteroaromatic moiety                or six-membered cyclic aromatic or heteroaromatic moiety                optionally substituted with hydroxyl groups; wherein                each carbon atom in R₅ individually comprises no more                than one hydroxyl group or no more than one ester or                carboxylic acid group; wherein R₅ optionally comprises                one or more ether linkages;            -   m=an integer ranging from 1 to the number of carbon                atoms in R₅; and wherein said esters have solubility in                water of at least 5 ppm at 25° C.;

        -   (ii) one or more glycerides having the structure

-   -   -   -   wherein R₁=a C1 to C21 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O);

        -   (iii) one or more esters of the formula:

-   -   -   -   wherein R₁=a C1 to C7 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂=a C1 to C10 straight chain or                branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl,                alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or                (CH₂CH(CH₃)—O)_(n)H and n is 1 to 10;

        -   (iv) one or more acetylated monosaccharides, acetylated            disaccharides, or acetylated polysaccharides; and

        -   (v) any combination of (i) through (iv);

    -   (2) a source of peroxygen; and

    -   (3) an enzyme catalyst comprising a polypeptide having        perhydrolytic activity and an amino acid sequence having at        least 80% identity to the amino acid sequence set forth in SEQ        ID NO: 8, provided that the amino acid residue bound to the        C-terminal side of the catalytic histidine is not glutamic acid;        -   whereby a peracid is enzymatically produced upon combining            the reaction components under suitable reaction conditions.

In another embodiment, a process for producing a peroxycarboxylic acidis also provided comprising:

-   -   (a) providing a set of reaction components comprising:        -   (1) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆=a C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆═C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety or a five-membered cyclic                    heteroaromatic moiety or six-membered cyclic                    aromatic or heteroaromatic moiety optionally                    substituted with hydroxyl groups; wherein each                    carbon atom in R₅ individually comprises no more                    than one hydroxyl group or no more than one ester or                    carboxylic acid group; wherein R₅ optionally                    comprises one or more ether linkages;                -   m=an integer ranging from 1 to the number of carbon                    atoms in R₅; and wherein said esters have solubility                    in water of at least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁=a C1 to C21 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₃ and R₄ are                    individually H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁=a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂=a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acylated monosaccharides, acylated                disaccharides, or acylated polysaccharides; and

            -   (v) any combination of (i) through (iv);

        -   (2) a source of peroxygen; and

        -   (3) an enzyme catalyst comprising a polypeptide having            perhydrolytic activity and an amino acid sequence having at            least 80% identity to the amino acid sequence set forth in            SEQ ID NO: 8, provided that the amino acid residue bound to            the C-terminal side of the catalytic histidine is not            glutamic acid;

    -   (b) combining the set of reaction components under suitable        reaction conditions whereby peroxycarboxylic acid is produced;        and

    -   (c) optionally diluting the peroxycarboxylic acid produced in        step (b).

In another embodiment, a process is provided further comprising a step(d) wherein the peroxycarboxylic acid produced in step (b) or step (c)is contacted with a hard surface, a body surface, or at least one anarticle of clothing.

The present process produces the desired peroxycarboxylic acid uponcombining the reaction components. The reaction components may remainseparated until use.

In a further aspect, a peroxycarboxylic acid generation and deliverysystem is provided comprising:

-   -   (a) a first compartment comprising        -   (1) an enzyme catalyst comprising a polypeptide having            perhydrolytic activity and an amino acid sequence having at            least 80% identity to the amino acid sequence set forth in            SEQ ID NO: 8 provided that the amino acid residue bound to            the C-terminal side of the catalytic histidine is not            glutamic acid;        -   (2) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆=a C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆═C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety or a five-membered cyclic                    heteroaromatic moiety or six-membered cyclic                    aromatic or heteroaromatic moiety optionally                    substituted with hydroxyl groups; wherein each                    carbon atom in R₅ individually comprises no more                    than one hydroxyl group or no more than one ester                    group or carboxylic acid group; wherein R₅                    optionally comprises one or more ether linkages;                -   m=an integer ranging from 1 to the number of carbon                    atoms in R₅; and                -   wherein said esters have solubility in water of at                    least 5 ppm at 25° C.; PP-25,C

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁=a C1 to C21 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₃ and R₄ are                    individually H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁=a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂=a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acylated monosaccharides, acylated                disaccharides, or acylated polysaccharides; and

            -   (v) any combination of (i) through (iv); and

        -   (3) an optional buffer; and

    -   (b) a second compartment comprising        -   (1) source of peroxygen;        -   (2) a peroxide stabilizer; and        -   (3) an optional buffer.

In a further embodiment, a laundry care composition is providedcomprising

-   -   a) a polypeptide having perhydrolytic activity and an amino acid        sequence having at least 80% identity to the amino acid sequence        set forth in SEQ ID NO: 8, provided that the amino acid residue        bound to the C-terminal side of the catalytic histidine is not        glutamic acid;    -   b) at least one substrate selected from the group consisting of:        -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   wherein            -   X=an ester group of the formula R₆—C(O)O;            -   R₆=a C1 to C7 linear, branched or cyclic hydrocarbyl                moiety, optionally substituted with hydroxyl groups or                C1 to C4 alkoxy groups, wherein R₆ optionally comprises                one or more ether linkages for R₆═C2 to C7;            -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl                moiety or a five-membered cyclic heteroaromatic moiety                or six-membered cyclic aromatic or heteroaromatic moiety                optionally substituted with hydroxyl groups; wherein                each carbon atom in R₅ individually comprises no more                than one hydroxyl group or no more than one ester group                or carboxylic acid group; wherein R₅ optionally                comprises one or more ether linkages;            -   m=an integer ranging from 1 to the number of carbon                atoms in R₅; and            -   wherein said esters have solubility in water of at least                5 ppm at 25° C.;

        -   (ii) one or more glycerides having the structure

-   -   -   -   wherein R₁=a C1 to C21 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O);

        -   (iii) one or more esters of the formula:

-   -   -   -   wherein R₁=a C1 to C7 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂=a C1 to C10 straight chain or                branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl,                alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or                (CH₂CH(CH₃)—O)_(n)H and n is 1 to 10;            -   (iv) one or more acylated monosaccharides, acylated                disaccharides, or acylated polysaccharides; and            -   (v) any combination of (i) through (iv); and

    -   c) a source of peroxygen; and

    -   d) at least one surfactant.

In a further embodiment, a personal care product is provided comprisinga polypeptide having perhydrolytic activity, said polypeptide having anamino acid sequence having at least 80% identity to the amino acidsequence set forth in SEQ ID NO: 8, provided that the amino acid residuebound to the C-terminal side of the catalytic histidine is not glutamicacid.

In a further embodiment, the personal care product is a shampoo, a bodylotion, a shower gel, a topical moisturizer, a toothpaste, a toothgel, amouthwash, a mouthrinse, an anti-plaque rinse or a topical cleanser.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1A-1B is a CLUSTALW alignment of SEQ ID NOs: 2, 4, 6, 8, 10, and12. Underlining indicates the three conserved submotifs of the CE-7esterase signature motif.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EuropeanPatent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules5.2 and 49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleic acid sequence of the codon-optimized codingregion encoding the Thermotoga maritima acetyl xylan esterase havingperhydrolytic activity.

SEQ ID NO: 2 is the amino acid sequence of the Thermotoga maritimaacetyl xylan esterase having perhydrolytic activity.

SEQ ID NO: 3 is the nucleic acid sequence of the codon-optimized codingregion encoding an Actinosynnema mirum acetyl xylan esterase havingperhydrolytic activity.

SEQ ID NO: 4 is the amino acid sequence of an Actinosynnema mirum acetylxylan esterase having perhydrolytic activity.

SEQ ID NO: 5 is the nucleic acid sequence of the codon-optimized codingregion encoding a Propionibacterium acnes acetyl xylan esterase havingperhydrolytic activity.

SEQ ID NO: 6 is the amino acid sequence of a Propionibacterium acnesacetyl xylan esterase having perhydrolytic activity.

SEQ ID NO: 7 is the nucleic acid sequence of the codon-optimized codingregion encoding a Streptococcus equi acetyl xylan esterase havingperhydrolytic activity.

SEQ ID NO: 8 is the amino acid sequence of a Streptococcus equi acetylxylan esterase having perhydrolytic activity.

SEQ ID NO: 9 is the nucleic acid sequence of the codon-optimized codingregion encoding a Stackebrandtia nassauensis acetyl xylan esterasehaving perhydrolytic activity.

SEQ ID NO: 10 is the amino acid sequence of a Stackebrandtia nassauensisacetyl xylan esterase having perhydrolytic activity.

SEQ ID NO: 11 is the nucleic acid sequence of the codon-optimized codingregion encoding a Streptococcus agalactiae acetyl xylan esterase havingperhydrolytic activity.

SEQ ID NO: 12 is the amino acid sequence of a Streptococcus agalactiaeacetyl xylan esterase having perhydrolytic activity.

SEQ ID NO: 13 is the nucleic acid sequence of the codon-optimized codingregion encoding an Actinosynnema mirum C277S variant acetyl xylanesterase having perhydrolytic activity.

SEQ ID NO: 14 is the amino acid sequence of an Actinosynnema mirum C277Svariant acetyl xylan esterase having perhydrolytic activity.

SEQ ID NO: 15 is the nucleic acid sequence of the codon-optimized codingregion encoding an Actinosynnema mirum C277T variant acetyl xylanesterase having perhydrolytic activity.

SEQ ID NO: 16 is the amino acid sequence of an Actinosynnema mirum C277Tvariant acetyl xylan esterase having perhydrolytic activity.

SEQ ID NO: 17 is the amino acid sequence of the Thermotoga maritimavariant C277S (U.S. Pat. No. 8,062,875).

SEQ ID NO: 18 is the amino acid sequence of the Thermotoga maritimavariant C277T (U.S. Pat. No. 8,062,875).

DETAILED DESCRIPTION

Compositions and methods are provided comprising a polypeptide havingperhydrolytic activity, the polypeptide having an amino acid sequencehaving at least 80% identity to the amino acid sequence set forth in SEQID NO: 8, provided that the amino acid residue bound to the C-terminalside of the catalytic histidine is not glutamic acid. The compositionsand methods are suitable to enzymatically produce at least one peracidsuitable for use in a laundry care product, a disinfectant product, acosmetic product or a personal care product.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an” and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

The term “comprising” means the presence of the stated features,integers, steps, or components as referred to in the claims, but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof. The term “comprising” isintended to include embodiments encompassed by the terms “consistingessentially of” and “consisting of”. Similarly, the term “consistingessentially of” is intended to include embodiments encompassed by theterm “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the term “multi-component system” will refer to a systemof enzymatically generating peroxycarboxylic acid wherein the componentsremain separated until use. As such, the multi-component system willinclude at least one first component that remains separated from atleast one second component. The first and second components areseparated in different compartments until use (i.e., using first andsecond compartments). The design of the multi-component systems willoften depend on the physical form of the components to be combined andare described in more detail below.

As used herein, the term “peroxycarboxylic acid” is synonymous withperacid, peroxyacid, peroxy acid, percarboxylic acid and peroxoic acid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate;1,2,3-triacetoxypropane; 1,2,3-propanetriol triacetate; and all othersynonyms of CAS Registry Number 102-76-1.

As used herein, the term “monobutyrin” is synonymous with glycerolmonobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term “dibutyrin” is synonymous with glyceroldibutyrate and glyceryl dibutyrate.

As used herein, the term “tributyrin” is synonymous with glyceroltributyrate; 1,2,3-tributyrylglycerol; and all other synonyms of CASRegistry Number 60-01-5.

As used herein, the term “monopropionin” is synonymous with glycerolmonopropionate, glycerin monopropionate, and glyceryl monopropionate.

As used herein, the term “dipropionin” is synonymous with glyceroldipropionate and glyceryl dipropionate.

As used herein, the term “tripropionin” is synonymous with glyceryltripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, andall other synonyms of CAS Registry Number 139-45-7.

As used herein, the terms “acylated sugar” and “acylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acylgroup, where the acyl group is selected from the group consisting ofstraight chain aliphatic carboxylates having a chain length from C2 toC8. Examples include, but are not limited to, glucose pentaacetate,xylose tetraacetate, acetylated xylan, acetylated xylan fragments,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-glucal.

As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and“hydrocarbyl moiety” mean a straight chain, branched or cyclicarrangement of carbon atoms connected by single, double, or triplecarbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In one embodiment, the hydrocarbyl moiety is astraight chain, branched or cyclic arrangement of carbon atoms connectedby single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms.

As used herein, the term “aromatic” refers to an organic compound ormoiety characterized by increased chemical stability resulting from thedelocalization of electrons in a ring system containing usually multipleconjugated double bonds. Planar monocyclic conjugated rings havingdelocalized electrons should be aromatic if the have (4n+2) π electrons.Examples of aromatic compounds may include derivatives of benzene (suchas 2-, 3- or 4-acetoxybenzoic acid). In one embodiment, the estersubstrate may be 4-acetoxybenzoic acid.

As used herein, the term “heterocyclic” refers to an organic compound ormoiety with a ring structure having one or more atoms other than carbonin at least one of its rings.

As used herein, the term “heteroaromatic” refers to an organic compoundor moiety with a ring structure that is both heterocyclic and aromatic,wherein the ring comprises at least one of the heteroatoms oxygen,nitrogen, or sulfur.

Examples of heteroaromatic moieties may include pyridine, pyrrole,furan, and thiophene moieties.

As used herein, the terms “monoesters” and “diesters” of 1,2-ethanediol,1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol,2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 2,5-pentanediol,1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol, 1,6-hexanediol, referto said compounds comprising at least one ester group of the formulaRC(O)O, wherein R is a C1 to C7 linear hydrocarbyl moiety.

As used herein, the terms “suitable enzymatic reaction formulation”,“components suitable for generation of a peroxycarboxylic acid”,“suitable reaction components”, “reaction components”, “reactionformulation”, and “suitable aqueous reaction formulation” refer to thematerials and water in which the reactants and the enzyme catalystcomprising the present variant polypeptide having perhydrolytic activitycome into contact to form the desired peroxycarboxylic acid. Thecomponents of the reaction formulation are provided herein and thoseskilled in the art appreciate the range of component variations suitablefor this process. In one embodiment, the enzymatic reaction formulationproduces peroxycarboxylic acid in situ upon combining the reactioncomponents. As such, the reaction components may be provided as amulti-component system wherein one or more of the reaction componentsremains separated until use. The design of systems and means forseparating and combining multiple active components are known in the artand generally will depend upon the physical form of the individualreaction components. For example, multiple active fluids (liquid-liquid)systems typically use multi-chamber dispenser bottles or two-phasesystems (U.S. Patent Application Publication No. 2005-0139608; U.S. Pat.No. 5,398,846; U.S. Pat. No. 5,624,634; U.S. Pat. No. 6,391,840; E.P.Patent 0807156B1; U.S. Patent Application Publication No. 2005-0008526;and PCT Publication No. WO 00/61713) such as found in some bleachingapplications wherein the desired bleaching agent is produced upon mixingthe reactive fluids. Multi-component formulations and multi-componentgeneration systems to enzymatically produce peroxycarboxylic acids fromcarboxylic acid esters are described by DiCosimo et al. in Published U.SPatent Application Nos. 2010-0086510 and 2010-0086621, respectively.Other forms of multi-component systems used to generate peroxycarboxylicacid may include, but are not limited to, those designed for one or moresolid components or combinations of solid-liquid components, such aspowders used in many commercially available bleaching compositions(e.g., U.S. Pat. No. 5,116,575), multi-layered tablets (e.g., U.S. Pat.No. 6,210,639), water dissolvable packets having multiple compartments(e.g., U.S. Pat. No. 6,995,125) and solid agglomerates that react uponthe addition of water (e.g., U.S. Pat. No. 6,319,888).

As used herein, the term “substrate” or “carboxylic acid estersubstrate” will refer to the reaction components enzymaticallyperhydrolyzed using the present enzyme catalyst in the presence of asuitable source of peroxygen, such as hydrogen peroxide. In oneembodiment, the substrate comprises at least one ester group capable ofbeing enzymatically perhydrolyzed using the enzyme catalyst, whereby aperoxycarboxylic acid is produced.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with a source of hydrogen peroxide to form aperoxycarboxylic acid. Typically, inorganic peroxide is reacted with theselected substrate in the presence of a catalyst to produce theperoxycarboxylic acid. As used herein, the term “chemical perhydrolysis”includes perhydrolysis reactions in which a substrate (such as aperoxycarboxylic acid precursor) is combined with a source of hydrogenperoxide wherein peroxycarboxylic acid is formed in the absence of anenzyme catalyst. As used herein, the term “enzymatic perhydrolysis”refers a reaction of a selected substrate with a source of hydrogenperoxide to form a peroxycarboxylic acid, wherein the reaction iscatalyzed by an enzyme catalyst having perhydrolysis activity.

As used herein, the term “perhydrolase activity” refers to the enzymecatalyst activity per unit mass (for example, milligram) of protein, drycell weight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmol of peroxycarboxylic acid product (such asperacetic acid) per minute at a specified temperature. “One unit ofenzyme activity” may also be used herein to refer to the amount ofperoxycarboxylic acid hydrolysis activity required for the hydrolysis of1 μmol of peroxycarboxylic acid (e.g., peracetic acid) per minute at aspecified temperature.

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme (i.e., a polypeptide) havingperhydrolysis activity and may be in the form of a whole microbial cell,permeabilized microbial cell(s), one or more cell components of amicrobial cell extract, partially purified enzyme, or purified enzyme.The enzyme catalyst may also be chemically modified (for example, bypegylation or by reaction with cross-linking reagents). The perhydrolasecatalyst may also be immobilized on a soluble or insoluble support usingmethods well-known to those skilled in the art; see for example,Immobilization of Enzymes and Cells; (2^(nd) Edition) Jose M. Guisan,Editor; Humana Press, Totowa, N.J., USA; 2006.

As used herein, “structurally classified as a CE-7 enzyme”,“structurally classified as a carbohydrate esterase family 7 enzyme”,“structurally classified as a CE-7 carbohydrate esterase”, and “CE-7perhydrolase” will be used herein to refer to enzymes havingperhydrolysis activity that are structurally classified as a CE-7carbohydrate esterase (see Cantarel et al., “The Carbohydrate-ActiveEnZymes database (CAZy): an expert resource for Glycogenomics”, NAR,37:D233-D238 (2009)).

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refer to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., Appl. Environ.Microbiol., 61(6): 2224-2229 (1995); U.S. Pat. No. 5,528,152; and U.S.Pat. No. 5,338,676).

As used herein, “acetyl xylan esterase” refers to an enzyme (E.C.3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylansand other acetylated saccharides.

As used herein, the term “Thermotoga maritima” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®NP_(—)227893.1). In one aspect, the Thermotoga maritima strain isThermotoga maritima MSB8. The amino acid sequence of the wild-typeenzyme having perhydrolase activity from Thermotoga maritima is providedas SEQ ID NO: 2.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid (or asdefined herein) Xaa X

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to, microorganisms, spores, viruses, prions, and mixturesthereof. The present enzyme can be used to produce an efficaciousconcentration of at least one peroxycarboxylic acid useful to reduceand/or eliminate the presence of the viable biological contaminants. Ina preferred embodiment, the biological contaminant is a viablepathogenic microorganism.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to an agent thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. Typically, disinfectants are used to treatinanimate objects or surfaces. As used herein, the term “antiseptic”refers to a chemical agent that inhibits the growth of disease-carryingmicroorganisms. In one aspect of the embodiment, the biologicalcontaminants are pathogenic microorganisms.

As used herein, the term “sanitary” means of or relating to therestoration or preservation of health, typically by removing, preventingor controlling an agent that may be injurious to health. As used herein,the term “sanitize” means to make sanitary. As used herein, the term“sanitizer” refers to a sanitizing agent. As used herein the term“sanitization” refers to the act or process of sanitizing.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses, and is synonymous with “viricide”. An agent thatexhibits the ability to inhibit or destroy viruses is described ashaving “virucidal” activity. Peroxycarboxylic acids can have virucidalactivity. Typical alternative virucides known in the art which may besuitable for use with the present invention include, for example,alcohols, ethers, chloroform, formaldehyde, phenols, beta propiolactone,iodine, chlorine, mercury salts, hydroxylamine, ethylene oxide, ethyleneglycol, quaternary ammonium compounds, enzymes, and detergents.

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peroxycarboxylic acids canhave biocidal activity. Typical alternative biocides known in the art,which may be suitable for use in the present invention include, forexample, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites,ozone, acrolein, amines, chlorinated phenolics, copper salts,organo-sulphur compounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viablemicroorganisms after treatment is at least a 3−log₁₀ reduction, morepreferably at least a 4−log₁₀ reduction, and most preferably at least a5−log₁₀ reduction. In another aspect, the minimum biocidal concentrationis at least a 6−log₁₀ reduction in viable microbial cells.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to, hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates, such as sodium percarbonate. As describedherein, the concentration of the hydrogen peroxide provided by theperoxygen compound in the aqueous reaction formulation is initially atleast 1 mM or more upon combining the reaction components. In oneembodiment, the hydrogen peroxide concentration in the aqueous reactionformulation is at least 0.5 mM. In another embodiment, the hydrogenperoxide concentration in the aqueous reaction formulation is at least10 mM. In another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is at least 100 mM. In another embodiment,the hydrogen peroxide concentration in the aqueous reaction formulationis at least 200 mM. In another embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is 500 mM or more. Inyet another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is 1000 mM or more. The molar ratio of thehydrogen peroxide to enzyme substrate, such as triglyceride,(H₂O₂:substrate) in the aqueous reaction formulation may be from about0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5to 5.

As used herein, the term “benefit agent” refers to a material thatpromotes or enhances a useful advantage, a favorable/desirable effect orbenefit. In one embodiment, a process is provided whereby a benefitagent, such as a composition comprising a peroxycarboxylic acid, isapplied to a textile or article of clothing to achieve a desiredbenefit, such as disinfecting, bleaching, destaining, deodorizing, andany combination thereof. In another embodiment, the present variantpolypeptide having perhydrolytic activity may be used to produce aperacid-based benefit agent for use in personal care products (such ashair care products, skin care products, nail care products or oral careproducts). In one embodiment, a personal care product is providedcomprising a polypeptide having perhydrolytic activity, said polypeptidehaving an amino acid sequence having at least 80% identity to the aminoacid sequence set forth in SEQ ID NO: 8, provided that the amino acidresidue bound to the C-terminal side of the catalytic histidine is notglutamic acid. The personal care products are formulated to provide asafe and efficacious concentration of the desired peracid benefit agent.

As used herein, “personal care products” means products used in thecleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth,including, but not limited to, shampoos, body lotions, shower gels,topical moisturizers, toothpaste, toothgels, mouthwashes, mouthrinses,anti-plaque rinses, and/or other topical cleansers. In some particularlypreferred embodiments, these products are utilized on humans, while inother embodiments, these products find use with non-human animals (e.g.,in veterinary applications).

As used herein, the terms “tooth whitening” and “tooth bleaching” areused interchangeably, to refer to improving the brightness (e.g.,whitening) of a tooth or teeth. It is intended that the term encompassany method suitable for whitening teeth, including the presentinvention, as well as chemical treatment, mild acid treatment, abrasivetooth whitening, and laser tooth whitening. In particularly preferredembodiments, the present invention provides a perhydrolase andperhydrolase-containing compositions suitable for whitening teeth.

Polypeptides Having Perhydrolytic Activity

The “signature motif” for CE-7 esterases previously reported to haveperhydrolytic activity is comprised three conserved submotifs (residueposition numbering relative to reference sequence SEQ ID NO: 2; thewild-type Thermotoga maritima acetyl xylan esterase):

a) Arg118-Gly119-Gln120; (“RGQ motif”);

b) Gly186-Xaa187-Ser188-Gln189-Gly190; and (“GXSQG motif”); and

c) His303-Glu304. (“HE motif”).

Typically, the Xaa at amino acid residue position 187 is glycine,alanine, proline, tryptophan, or threonine. Two of the three amino acidresidues belonging to the catalytic triad are in bold.

Although the present perhydrolytic enzymes contain the RGQ motif and theGXSQG motif, none of the present perhydrolytic enzymes contain theglutamic acid within the “HE motif” previously reported as a conservedstructural motif as shown in Table A and FIG. 1.

TABLE A Motifs found within the present enzymes having perhydrolaseactivity. Amino acid GXSQG HX residue bound RGQ motif motif motif tocatalytic Perhydrolase (Residue (Residue (Residue histidine Sequence #s)#s) #s) in HX motif SEQ ID NO: 2^(a) 118-120 186-190 303-304  E^(b) SEQID NO: 4 118-120 184-188 302-303 A SEQ ID NO: 6 117-119 184-188 309-310A SEQ ID NO: 8 114-116 177-181 299-300 D SEQ ID NO: 10 117-119 186-190303-304 S SEQ ID NO: 12 115-117 178-182 299-300 D SEQ ID NO: 14 118-120184-188 302-303 A SEQ ID NO: 16 118-120 184-188 302-303 A ^(a)=Thermotoga maritima reference sequence. ^(b)= Previously reported to bea conserved glutamic acid, forming an “HE” motif.

It appears the present polypeptides having perhydrolytic activity mayrepresent a new subgroup within the larger generic class of CE-7carbohydrate esterases listed as members within the CAZy database(Cantarel et al., “The Carbohydrate-Active EnZymes database (CAZy): anexpert resource for Glycogenomics”, NAR, 37:D233-D238 (2009)). As such,the polypeptides having perhydrolytic activity used within the presentapplication will be referred to herein as “CE-7 carbohydrate esterases”or “CE-7 perhydrolases” even though they may lack a portion of thepreviously defined “signature motif”.

In another embodiment, the present polypeptides having perhydrolyticactivity are further defined as having the following combination ofmotifs when aligned against reference sequence SEQ ID NO: 2 (residueposition numbering relative to reference sequence SEQ ID NO: 2; thewild-type Thermotoga maritima acetyl xylan esterase):

a) Arg118-Gly119-Gln120; (“RGQ motif”);

b) Gly186-Xaa187-Ser188-Gln189-Gly190; and (“GXSQG motif”); and

c) His303-Xaa304. (“HX motif”); wherein “Xaa” is not glutamic acid.

In a preferred aspect, “X” amino acid residue within the “HX motif” isalanine, aspartic acid, or serine.

In another aspect, the present polypeptide having perhydrolytic activitycomprises an amino acid sequence having at least 80% identity to anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,6, 8, 10, 12, 14, and 16, provided that the amino acid residue bound tothe C-terminal side of the catalytic histidine is not glutamic acid.

In one embodiment, the present polypeptides having perhydrolyticactivity have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% amino acid identity to the sequences provided herein, providedthat the amino acid residue bound to the C-terminal side of thecatalytic histidine is not glutamic acid.

In another aspect, the present polypeptide having perhydrolytic activitycomprises an amino acid sequence having at least 80% identity to anamino acid sequence having at least 80% identity to the amino acidsequence set forth in SEQ ID NO: 8, provided that the amino acid residuebound to the C-terminal side of the catalytic histidine is not glutamicacid. In another aspect, the present polypeptide having perhydrolyticactivity comprises an amino acid sequence SEQ ID NO: 8.

As used herein, the term “variant perhydrolase” or “variant” will referto perhydrolytic enzymes having a modification that results in at leastone amino acid addition, deletion, and/or substitution when compared tothe corresponding enzyme (typically the wild type enzyme) from which thevariant was derived; so long as the requisite motifs described hereinand the associated perhydrolytic activity are maintained. CE-7 variantperhydrolases may also be used in the present compositions and methods.Examples of variants are provided as SEQ ID NOs: 14 and 16.

The skilled artisan recognizes that substantially similar perhydrolasesequences may also be used in the present compositions and methods. Inone embodiment, substantially similar sequences are defined by theirability to hybridize, under highly stringent conditions, with thenucleic acid molecules associated with sequences exemplified herein. Inanother embodiment, sequence alignment algorithms may be used to definesubstantially similar enzymes based on the percent identity to the DNAor amino acid sequences provided herein.

As used herein, a nucleic acid molecule is “hybridizable” to anothernucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when asingle strand of the first molecule can anneal to the other moleculeunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known and exemplified inSambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(2001). The conditions of temperature and ionic strength determine the“stringency” of the hybridization. Stringency conditions can be adjustedto screen for moderately similar molecules, such as homologous sequencesfrom distantly related organisms, to highly similar molecules, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes typically determine stringency conditions. Oneset of preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A more preferred set of conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringenthybridization conditions is 0.1×SSC, 0.1% SDS, 65° C. and washed with2×SSC, 0.1% SDS followed by a final wash of 0.1×SSC, 0.1% SDS, 65° C.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (Sambrook andRussell, supra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrookand Russell, supra). In one aspect, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Preferably, a minimumlength for a hybridizable nucleic acid is at least about 15 nucleotidesin length, more preferably at least about 20 nucleotides in length, evenmore preferably at least 30 nucleotides in length, even more preferablyat least 300 nucleotides in length, and most preferably at least 800nucleotides in length. Furthermore, the skilled artisan will recognizethat the temperature and wash solution salt concentration may beadjusted as necessary according to factors such as length of the probe.

As used herein, the term “percent identity” is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.), the AlignX program of Vector NTI v. 7.0 (Informax, Inc.,Bethesda, Md.), or the EMBOSS Open Software Suite (EMBL-EBI; Rice etal., Trends in Genetics 16, (6):276-277 (2000)). Multiple alignment ofthe sequences can be performed using the CLUSTAL method (such asCLUSTALW; for example version 1.83) of alignment (Higgins and Sharp,CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res.22:4673-4680 (1994); and Chema et al., Nucleic Acids Res 31(13):3497-500 (2003)), available from the European Molecular BiologyLaboratory via the European Bioinformatics Institute) with the defaultparameters. Suitable parameters for CLUSTALW protein alignments includeGAP Existence penalty=15, GAP extension=0.2, matrix=Gonnet (e.g.,Gonnet250), protein ENDGAP=−1, protein GAPDIST=4, and KTUPLE=1. In oneembodiment, a fast or slow alignment is used with the default settingswhere a slow alignment is preferred. Alternatively, the parameters usingthe CLUSTALW method (e.g., version 1.83) may be modified to also useKTUPLE=1, GAP PENALTY=10, GAP extension=1, matrix=BLOSUM (e.g.,BLOSUM64), WINDOW=5, and TOP DIAGONALS SAVED=5.

By “catalytic histidine” is meant the histidine residue in the presentlydisclosed perhydrolases that forms a catalytic triad with serine andaspartic acid. For example, in SEQ ID NO: 8, the catalytic histidine isamino acid residue number 299. A variant of SEQ ID NO: 8 that hasperhydrolase activity will have its catalytic histidine align with thecatalytic histidine of SEQ ID NO: 8 when the sequences are comparedusing CLUSTALW, meaning that the variant's catalytic histidine may, butdoes not have to be, at amino acid position 299 of the variant.

In one aspect, suitable isolated nucleic acid molecules encode apolypeptide having an amino acid sequence that is at least 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 84, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acidsequences reported herein. Suitable nucleic acid molecules not only havethe above homologies, but also typically encode a polypeptide havingabout 210 to 340 amino acids in length, about 300 to about 340 aminoacids, preferably about 310 to about 330 amino acids, and mostpreferably about 318 to about 325 amino acids in length wherein eachpolypeptide is characterized as having perhydrolytic activity.

Suitable Reaction Conditions for the Enzyme-Catalyzed Preparation ofPeroxycarboxylic Acids from Carboxylic Acid Esters and Hydrogen Peroxide

A process is provided to produce an aqueous formulation comprising atleast one peroxycarboxylic acid by reacting carboxylic acid esters andan inorganic peroxide (such as hydrogen peroxide, sodium perborate orsodium percarbonate) in the presence of an enzyme catalyst havingperhydrolysis activity, wherein the enzyme catalyst comprises, in oneembodiment, a polypeptide having at least 80% identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10,12, 14, and 16, provided that the amino acid residue bound to theC-terminal side of the catalytic histidine is not glutamic acid. In afurther embodiment, the polypeptide having perhydrolytic activitycomprises an amino acid sequence selected from SEQ ID NOs: 4, 6, 8, 10,12, 14, and 16. In a further embodiment, the polypeptide has the aminoacid sequence of SEQ ID NO: 8.

In one embodiment, suitable substrates include one or more estersprovided by the following formula:

[X]_(m)R₅

-   -   wherein X=an ester group of the formula R₆C(O)O    -   R₆=a C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with hydroxyl groups or C1 to C4 alkoxy        groups, wherein R₆ optionally comprises one or more ether        linkages for R₆═C2 to C7;    -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or        a five-membered cyclic heteroaromatic moiety or six-membered        cyclic aromatic or heteroaromatic moiety optionally substituted        with hydroxyl groups; wherein each carbon atom in R₅        individually comprises no more than one hydroxyl group or no        more than one ester group or carboxylic acid group; wherein R₅        optionally comprises one or more ether linkages;    -   m=an integer ranging from 1 to the number of carbon atoms in R₅;        and    -   wherein said esters have solubility in water of at least 5 ppm        at 25° C.

In another embodiment, R₆=a C1 to C7 linear hydrocarbyl moiety,optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups,optionally comprising one or more ether linkages. In a further preferredembodiment, R₆=a C2 to C7 linear hydrocarbyl moiety, optionallysubstituted with hydroxyl groups, and/or optionally comprising one ormore ether linkages.

In one embodiment, the suitable substrate may include 2-acetoxybenzoicacid, 3-acetoxybenzoic acid, 4-acetoxybenzoic acid or mixtures thereof.

In another embodiment, suitable substrates also include one or moreglycerides of the formula:

wherein R₁=a C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O). In one embodiment, the suitable substrateis a glyceride of the above formula wherein R₁=a C1 to C7 straight chainor branched chain alkyl optionally substituted with an hydroxyl or a C1to C4 alkoxy group and R₃ and R₄ are individually H or R₁C(O).

In another aspect, suitable substrates may also include one or moreesters of the formula:

wherein R₁=a C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=a C1 toC10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10.

Suitable substrates may also include one or more acylated saccharidesselected from the group consisting of acylated mono-, di-, andpolysaccharides.

In another embodiment, the acylated saccharides are selected from thegroup consisting of acetylated xylan, fragments of acetylated xylan,acetylated xylose (such as xylose tetraacetate), acetylated glucose(such as α-D-glucose pentaacetate; β-D-glucose pentaacetate),β-D-galactose pentaacetate, sorbitol hexaacetate, sucrose octaacetate,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal,tri-O-acetyl-D-glucal, tetraacetylxylofuranose, α-D-glucopyranosepentaacetate, α-D-mannopyranose pentaacetate, and acetylated cellulose.In a preferred embodiment, the acetylated saccharide is selected fromthe group consisting of β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucal, sucrose octaacetate, andacetylated cellulose.

In another embodiment, suitable substrates are selected from the groupconsisting of: monoacetin; diacetin; triacetin; monopropionin;dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucosepentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylanfragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,2,5-pentanediol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol,1,6-hexanediol; and mixtures thereof.

In another embodiment, the carboxylic acid ester is selected from thegroup consisting of monoacetin, diacetin, triacetin, and combinationsthereof. In another embodiment, the substrate is a C1 to C6 polyolcomprising one or more ester groups. In a preferred embodiment, one ormore of the hydroxyl groups on the C1 to C6 polyol are substituted withone or more acetoxy groups (such as 1,3-propanediol diacetate,1,4-butanediol diacetate, etc.). In a further embodiment, the substrateis propylene glycol diacetate (PGDA), ethylene glycol diacetate (EGDA),or a mixture thereof.

In another embodiment, suitable substrates are selected from the groupconsisting of ethyl acetate; methyl lactate; ethyl lactate; methylglycolate; ethyl glycolate; methyl methoxyacetate; ethyl methoxyacetate;methyl 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; triethyl 2-acetylcitrate; glucose pentaacetate; gluconolactone; glycerides (mono-, di-,and triglycerides) such as monoacetin, diacetin, triacetin,monopropionin, dipropionin (glyceryl dipropionate), tripropionin(1,2,3-tripropionylglycerol), monobutyrin, dibutyrin (glyceryldibutyrate), tributyrin (1,2,3-tributyrylglycerol); acetylatedsaccharides; and mixtures thereof.

In a further embodiment, suitable substrates are selected from the groupconsisting of monoacetin, diacetin, triacetin, monopropionin,dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, ethylacetate, and ethyl lactate. In yet another aspect, the substrate isselected from the group consisting of diacetin, triacetin, ethylacetate, and ethyl lactate. In a most preferred embodiment, the suitablesubstrate comprises triacetin.

The carboxylic acid ester is present in the aqueous reaction formulationat a concentration sufficient to produce the desired concentration ofperoxycarboxylic acid upon enzyme-catalyzed perhydrolysis. Thecarboxylic acid ester need not be completely soluble in the aqueousreaction formulation, but has sufficient solubility to permit conversionof the ester by the perhydrolase catalyst to the correspondingperoxycarboxylic acid. The carboxylic acid ester is present in theaqueous reaction formulation at a concentration of 0.0005 wt % to 40 wt% of the aqueous reaction formulation, preferably at a concentration of0.01 wt % to 20 wt % of the aqueous reaction formulation, and morepreferably at a concentration of 0.05 wt % to 10 wt % of the aqueousreaction formulation. The wt % of carboxylic acid ester may optionallybe greater than the solubility limit of the carboxylic acid ester, suchthat the concentration of the carboxylic acid ester is at least 0.0005wt % in the aqueous reaction formulation that is comprised of water,enzyme catalyst, and source of peroxide, where the remainder of thecarboxylic acid ester remains as a second separate phase of a two-phaseaqueous/organic reaction formulation. Not all of the added carboxylicacid ester must immediately dissolve in the aqueous reactionformulation, and after an initial mixing of all reaction components,additional continuous or discontinuous mixing is optional.

The peroxycarboxylic acids produced by the present reaction componentsmay vary depending upon the selected substrates, so long as the presentenzyme catalyst is used. In one embodiment, the peroxycarboxylic acidproduced is peracetic acid, perpropionic acid, perbutyric acid,peroctanoic acid, perlactic acid, perglycolic acid, permethoxyaceticacid, per-β-hydroxybutyric acid, or mixtures thereof.

The peroxygen source may include, but is not limited to, hydrogenperoxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct(carbamide peroxide)), perborate salts and percarbonate salts.Alternatively, hydrogen peroxide can be generated in situ by thereaction of a substrate and oxygen catalyzed by an enzyme having oxidaseactivity (including, but not limited to, glucose oxidase, galactoseoxidase, sorbitol oxidase, hexose oxidase, alcohol oxidase, glyceroloxidase, monoamine oxidase, glycolate oxidase, lactate oxidase, pyruvateoxidase, oxalate oxidase, choline oxidase, cholesterol oxidase, pyranoseoxidase, carboxyalcohol oxidase, L-amino acid oxidase, glycine oxidase,glutamate oxidase, lysine oxidase, and uricase). The concentration ofperoxygen compound in the aqueous reaction formulation may range from0.0033 wt % to about 50 wt %, preferably from 0.033 wt % to about 40 wt%, more preferably from 0.33 wt % to about 30 wt %.

Many perhydrolase catalysts (such as whole cells, permeabilized wholecells, and partially purified whole cell extracts) have been reported tohave catalase activity (EC 1.11.1.6). Catalases catalyze the conversionof hydrogen peroxide into oxygen and water. In one aspect, the enzymecatalyst having perhydrolase activity lacks catalase activity. Inanother aspect, the enzyme catalyst having perhydrolase activity has asufficiently-low catalase activity that the presence of said catalaseactivity does not significantly interfere with perhydrolase-catalyzedperoxycarboxylic acid production. In another aspect, a catalaseinhibitor is added to the aqueous reaction formulation. Examples ofcatalase inhibitors include, but are not limited to, sodium azide andhydroxylamine sulfate. One of skill in the art can adjust theconcentration of catalase inhibitor as needed. The concentration of thecatalase inhibitor typically ranges from 0.1 mM to about 1 M; preferablyabout 1 mM to about 50 mM; more preferably from about 1 mM to about 20mM. In one aspect, sodium azide concentration typically ranges fromabout 20 mM to about 60 mM while hydroxylamine sulfate is concentrationis typically about 0.5 mM to about 30 mM, preferably about 10 mM.

The catalase activity in a host cell can be down-regulated or eliminatedby disrupting expression of the gene(s) responsible for the catalaseactivity using well known techniques including, but not limited to,transposon mutagenesis, RNA antisense expression, targeted mutagenesis,and random mutagenesis. In a preferred embodiment, the gene(s) encodingthe endogenous catalase activity are down-regulated or disrupted (i.e.,“knocked-out”). As used herein, a “disrupted” gene is one where theactivity and/or function of the protein encoded by the modified gene areno longer present. Means to disrupt a gene are well-known in the art andmay include, but are not limited to, insertions, deletions, or mutationsto the gene so long as the activity and/or function of the correspondingprotein is no longer present. In a further preferred embodiment, theproduction host is an E. coli production host comprising a disruptedcatalase gene selected from the group consisting of katG and katE (seeU.S. Pat. No. 7,951,566 to DiCosimo et al.). In another embodiment, theproduction host is an E. coli strain comprising a down-regulation and/ordisruption in both katG and katE catalase genes. An E. coli straincomprising a double-knockout of katG and katE has been prepared and isdescribed as E. coli strain KLP18 (U.S. Pat. No. 7,951,566 to DiCosimoet al.).

The concentration of the catalyst in the aqueous reaction formulationdepends on the specific catalytic activity of the catalyst, and ischosen to obtain the desired rate of reaction. The weight of catalyst inperhydrolysis reactions typically ranges from 0.0001 mg to 50 mg per mLof total reaction volume, preferably from 0.0005 mg to 10 mg per mL,more preferably from 0.0010 mg to 2.0 mg per mL. The catalyst may alsobe immobilized on a soluble or insoluble support using methodswell-known to those skilled in the art; see for example, Immobilizationof Enzymes and Cells (2^(nd) Edition); Jose M. Guisan, Editor; HumanaPress, Totowa, N.J., USA; 2006. The use of immobilized catalysts permitsthe recovery and reuse of the catalyst in subsequent reactions. Theenzyme catalyst may be in the form of whole microbial cells,permeabilized microbial cells, microbial cell extracts,partially-purified or purified enzymes, and mixtures thereof.

In one aspect, the concentration of peroxycarboxylic acid generated bythe combination of chemical perhydrolysis and enzymatic perhydrolysis ofthe carboxylic acid ester is sufficient to provide an effectiveconcentration of peroxycarboxylic acid for disinfection, bleaching,sanitization, deodorizing or destaining at a desired pH. In anotheraspect, the peroxycarboxylic acid is generated at a safe and efficaciousconcentration suitable for use in a personal care product to be appliedto the hair, skin, nails or tissues of the oral cavity, such as toothenamel, tooth pellicle or the gums. In another aspect, the presentmethods provide combinations of enzymes and enzyme substrates to producethe desired effective concentration of peroxycarboxylic acid, where, inthe absence of added enzyme, there is a significantly lowerconcentration of peroxycarboxylic acid produced. Although there may besome chemical perhydrolysis of the enzyme substrate by direct chemicalreaction of inorganic peroxide with the enzyme substrate, there may notbe a sufficient concentration of peroxycarboxylic acid generated toprovide an effective concentration of peroxycarboxylic acid in thedesired applications, and a significant increase in totalperoxycarboxylic acid concentration is achieved by the addition of anappropriate perhydrolase catalyst to the aqueous reaction formulation.

In one aspect of the invention, the concentration of peroxycarboxylicacid generated (e.g. peracetic acid) by the enzymatic perhydrolysis isat least about 2 ppm, preferably at least 20 ppm, preferably at least100 ppm, more preferably at least about 200 ppm peroxycarboxylic acid,more preferably at least 300 ppm, more preferably at least 500 ppm, morepreferably at least 700 ppm, more preferably at least about 1000 ppmperoxycarboxylic acid, more preferably at least about 2000 ppmperoxycarboxylic acid, most preferably at least 10,000 ppmperoxycarboxylic acid within 5 minutes more preferably within 1 minuteof initiating the enzymatic perhydrolysis reaction. In a second aspectof the invention, the concentration of peroxycarboxylic acid generated(e.g. peracetic acid) by the enzymatic perhydrolysis is at least about 2ppm, preferably at least 20 ppm, preferably at least 30 ppm, morepreferably at least about 40 ppm peroxycarboxylic acid, more preferablyat least 50 ppm, more preferably at least 60 ppm, more preferably atleast 70 ppm, more preferably at least about 80 ppm peroxycarboxylicacid, most preferably at least 100 ppm peroxycarboxylic acid within 5minutes, more preferably within 1 minute, of initiating the enzymaticperhydrolysis reaction (i.e., time measured from combining the reactioncomponents to form the formulation).

The aqueous formulation comprising the peroxycarboxylic acid may beoptionally diluted with diluent comprising water, or a solutionpredominantly comprised of water, to produce a formulation with thedesired lower target concentration of peroxycarboxylic acid. In oneaspect, the reaction time required to produce the desired concentration(or concentration range) of peroxycarboxylic acid is about 20 minutes orless, preferable about 5 minutes or less, most preferably about 1 minuteor less.

In other aspects, the surface or inanimate object contaminated with aconcentration of a biological contaminant(s) is contacted with theperoxycarboxylic acid formed in accordance with the processes describedherein within about 1 minute to about 168 hours of combining saidreaction components, or within about 1 minute to about 48 hours, orwithin about 1 minute to 2 hours of combining said reaction components,or any such time interval therein.

In another aspect, the peroxycarboxylic acid formed in accordance withthe processes describe herein is used in a laundry care applicationwherein the peroxycarboxylic acid is contacted with clothing or atextile to provide a benefit, such as disinfecting, bleaching,destaining, deodorizing and/or a combination thereof. Theperoxycarboxylic acid may be used in a variety of laundry care productsincluding, but not limited to, laundry or textile pre-wash treatments,laundry detergents or additives, stain removers, bleaching compositions,deodorizing compositions, and rinsing agents. In one embodiment, thepresent process to produce a peroxycarboxylic acid for a target surfaceis conducted in situ.

In the context of laundry care applications, the term “contacting anarticle of clothing or textile” means that the article of clothing ortextile is exposed to a formulation disclosed herein. To this end, thereare a number of formats the formulation may be used to treat articles ofclothing or textiles including, but not limited to, liquid, solids, gel,paste, bars, tablets, spray, foam, powder, or granules and can bedelivered via hand dosing, unit dosing, dosing from a substrate,spraying and automatic dosing from a laundry washing or drying machine.Granular compositions can also be in compact form; liquid compositionscan also be in a concentrated form.

When the formulations disclosed herein are used in a laundry washingmachine, the formulation can further contain components typical tolaundry detergents. For example, typical components include, but are notlimited to, surfactants, bleaching agents, bleach activators, additionalenzymes, suds suppressors, dispersants, lime-soap dispersants, soilsuspension and anti-redeposition agents, softening agents, corrosioninhibitors, tarnish inhibitors, germicides, pH adjusting agents,non-builder alkalinity sources, chelating agents, organic and/orinorganic fillers, solvents, hydrotropes, optical brighteners, dyes, andperfumes. The formulations disclosed herein can also be used asdetergent additive products in solid or liquid form. Such additiveproducts are intended to supplement or boost the performance ofconventional detergent compositions and can be added at any stage of thecleaning process.

In connection with the present systems and methods for laundry carewhere the peracid is generated for one or more of bleaching, stainremoval, and odor reduction, the concentration of peracid generated(e.g., peracetic acid) by the perhydrolysis of at least one carboxylicacid ester may be at least about 2 ppm, preferably at least 20 ppm,preferably at least 100 ppm, and more preferably at least about 200 ppmperacid. In connection with the present systems and methods for laundrycare where the peracid is generated for disinfection or sanitization,the concentration of peracid generated (e.g., peracetic acid) by theperhydrolysis of at least one carboxylic acid ester may be at leastabout 2 ppm, more preferably at least 20 ppm, more preferably at least200 ppm, more preferably at least 500 ppm, more preferably at least 700ppm, more preferably at least about 1000 ppm peracid, most preferably atleast 2000 ppm peracid within 10 minutes, preferably within 5 minutes,and most preferably within 1 minute of initiating the perhydrolysisreaction. The product formulation comprising the peracid may beoptionally diluted with water, or a solution predominantly comprised ofwater, to produce a formulation with the desired lower concentration ofperacid. In one aspect of the present methods and systems, the reactiontime required to produce the desired concentration of peracid is notgreater than about two hours, preferably not greater than about 30minutes, more preferably not greater than about 10 minutes, even morepreferably not greater than about 5 minutes, and most preferably inabout 1 minute or less.

The temperature of the reaction is chosen to control both the reactionrate and the stability of the enzyme catalyst activity. The temperatureof the reaction may range from just above the freezing point of theaqueous reaction formulation (approximately 0° C.) to about 85° C., witha preferred range of reaction temperature of from about 5° C. to about75° C.

The pH of the aqueous reaction formulation while enzymatically producingperoxycarboxylic acid is maintained at a pH ranging from about 5.0 toabout 10.0, preferably about 6.5 to about 8.5, and yet even morepreferably about 6.5 to about 7.5. In one embodiment, the pH of theaqueous reaction formulation ranges from about 6.5 to about 8.5 for atleast 30 minutes after combining the reaction components. The pH of theaqueous reaction formulation may be adjusted or controlled by theaddition or incorporation of a suitable buffer, including, but notlimited to, phosphate, pyrophosphate, bicarbonate, acetate, or citrate.In one embodiment, the buffer is selected from a phosphate buffer, abicarbonate buffer, or a buffer formed by the combination of hard water(tap water to simulate laundry care applications) and percarbonate (fromsodium percarbonate used to generate hydrogen peroxide). Theconcentration of buffer, when employed, is typically from 0.1 mM to 1.0M, preferably from 1 mM to 300 mM, most preferably from 10 mM to 100 mM.In another aspect of the present invention, no buffer is added to thereaction mixture while enzymatically producing peroxycarboxylic acid.

In yet another aspect, the enzymatic perhydrolysis aqueous reactionformulation may contain an organic solvent that acts as a dispersant toenhance the rate of dissolution of the carboxylic acid ester in theaqueous reaction formulation. Such solvents include, but are not limitedto, propylene glycol methyl ether, acetone, cyclohexanone, diethyleneglycol butyl ether, tripropylene glycol methyl ether, diethylene glycolmethyl ether, propylene glycol butyl ether, dipropylene glycol methylether, cyclohexanol, benzyl alcohol, isopropanol, ethanol, propyleneglycol, and mixtures thereof.

In another aspect, the enzymatic perhydrolysis product may containadditional components that provide desirable functionality. Theseadditional components include, but are not limited to, buffers,detergent builders, thickening agents, emulsifiers, surfactants, wettingagents, corrosion inhibitors (e.g., benzotriazole), enzyme stabilizers,and peroxide stabilizers (e.g., metal ion chelating agents). Many of theadditional components are well known in the detergent industry (see, forexample, U.S. Pat. No. 5,932,532; hereby incorporated by reference).Examples of emulsifiers include, but are not limited to, polyvinylalcohol or polyvinylpyrrolidone. Examples of thickening agents include,but are not limited to, LAPONITE® RD (synthetic layered silicate), cornstarch, PVP, CARBOWAX® (polyethylene glycol and/or methoxypolyethyleneglycol), CARBOPOL® (acrylates crosspolymer), CABOSIL® (syntheticamphormous fumed silicon dioxide), polysorbate 20, PVA, and lecithin.Examples of buffering systems include, but are not limited to, sodiumphosphate monobasic/sodium phosphate dibasic; sulfamicacid/triethanolamine; citric acid/triethanolamine; tartaricacid/triethanolamine; succinic acid/triethanolamine; and aceticacid/triethanolamine. Examples of surfactants include, but are notlimited to, a) non-ionic surfactants such as block copolymers ofethylene oxide or propylene oxide, ethoxylated or propoxylated linearand branched primary and secondary alcohols, and aliphatic phosphineoxides; b) cationic surfactants such as quaternary ammonium compounds,particularly quaternary ammonium compounds having a C8-C20 alkyl groupbound to a nitrogen atom additionally bound to three C1-C2 alkyl groups;c) anionic surfactants such as alkane carboxylic acids (e.g., C8-C20fatty acids), alkyl phosphonates, alkane sulfonates (e.g., sodiumdodecylsulphate “SDS”) or linear or branched alkyl benzene sulfonates,alkene sulfonates; and d) amphoteric and zwitterionic surfactants suchas aminocarboxylic acids, aminodicarboxylic acids, alkybetaines, andmixtures thereof. Additional components may include fragrances, dyes,stabilizers of hydrogen peroxide (e.g., metal chelators such as1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST® 2010, Solutia Inc.,St. Louis, Mo.) and ethylenediaminetetraacetic acid (EDTA)), TURPINAL®SL (etidronic acid), DEQUEST® 0520 (phosphonate), DEQUEST® 0531(phosphonate), stabilizers of enzyme activity (e.g., polyethylene glycol(PEG)), and detergent builders.

In another aspect, the enzymatic perhydrolysis product may be pre-mixedto generate the desired concentration of peroxycarboxylic acid prior tocontacting the surface or inanimate object to be disinfected.

In another aspect, the enzymatic perhydrolysis product is not pre-mixedto generate the desired concentration of peroxycarboxylic acid prior tocontacting the surface or inanimate object to be disinfected, butinstead, the components of the aqueous reaction formulation thatgenerate the desired concentration of peroxycarboxylic acid arecontacted with the surface or inanimate object to be disinfected and/orbleached or destained, generating the desired concentration ofperoxycarboxylic acid. In some embodiments, the components of theaqueous reaction formulation combine or mix at the locus. In someembodiments, the reaction components are delivered or applied to thelocus and subsequently mix or combine to generate the desiredconcentration of peroxycarboxylic acid.

Production of Peroxycarboxylic Acids Using a Perhydrolase Catalyst

The peroxycarboxylic acids, once produced, are quite reactive and maydecrease in concentration over extended periods of time, depending onvariables that include, but are not limited to, temperature and pH. Assuch, it may be desirable to keep the various reaction componentsseparated, especially for liquid formulations. In one aspect, thehydrogen peroxide source is separate from either the substrate or theperhydrolase catalyst, preferably from both. This can be accomplishedusing a variety of techniques including, but not limited to, the use ofmulticompartment chambered dispensers (U.S. Pat. No. 4,585,150) and atthe time of use physically combining the perhydrolase catalyst with asource of peroxygen (such as hydrogen peroxide) and the presentsubstrates to initiate the aqueous enzymatic perhydrolysis reaction. Theperhydrolase catalyst may optionally be immobilized within the body ofreaction chamber or separated (e.g., filtered, etc.) from the reactionproduct comprising the peroxycarboxylic acid prior to contacting thesurface and/or object targeted for treatment. The perhydrolase catalystmay be in a liquid matrix or in a solid form (e.g., powder or tablet) orembedded within a solid matrix that is subsequently mixed with thesubstrates to initiate the enzymatic perhydrolysis reaction. In afurther aspect, the perhydrolase catalyst may be contained within adissolvable or porous pouch that may be added to the aqueous substratematrix to initiate enzymatic perhydrolysis. In yet a further aspect, theperhydrolase catalyst may comprise the contents contained within aseparate compartment of a dissolvable or porous pouch that has at leastone additional compartment for the containment contents comprising theester substrate and/or source of peroxide. In an additional furtheraspect, a powder comprising the enzyme catalyst is suspended in thesubstrate (e.g., triacetin), and at time of use is mixed with a sourceof peroxygen in water.

Method for Determining the Concentration of Peroxycarboxylic Acid andHydrogen Peroxide.

A variety of analytical methods can be used in the present method toanalyze the reactants and products including, but not limited to,titration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), capillary electrophoresis(CE), the HPLC analytical procedure described by U. Karst et al. (Anal.Chem., 69(17):3623-3627 (1997)), and the2,2′-azino-bis(3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (see U.Pinkernell et al., The Analyst 122:567-571 (1997); S. Minning, et al.,Analytica Chimica Acta 378:293-298 (1999) and WO 2004/058961 A1) asdescribed in U.S. Pat. No. 7,951,566.

Determination of Minimum Biocidal Concentration of PeroxycarboxylicAcids

The method described by J. Gabrielson et al. (J. Microbiol. Methods 50:63-73 (2002)) can be employed for determination of the Minimum BiocidalConcentration (MBC) of peroxycarboxylic acids, or of hydrogen peroxideand enzyme substrates. The assay method is based on XTT reductioninhibition, where XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to, viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Uses of Enzymatically Prepared Peroxycarboxylic Acid Compositions

The enzyme catalyst-generated peroxycarboxylic acid produced accordingto the present method can be used in a variety of hard surface/inanimateobject applications for reduction of concentrations of biologicalcontaminants, such as decontamination of medical instruments (e.g.,endoscopes), textiles (such as garments and carpets), food preparationsurfaces, food storage and food-packaging equipment, materials used forthe packaging of food products, chicken hatcheries and grow-outfacilities, animal enclosures, and spent process waters that havemicrobial and/or virucidal activity. The enzyme-generatedperoxycarboxylic acids may be used in formulations designed toinactivate prions (e.g., certain proteases) to additionally providebiocidal activity (see U.S. Pat. No. 7,550,420 to DiCosimo et al.).

In one aspect, the peroxycarboxylic acid composition is useful as adisinfecting agent for non-autoclavable medical instruments and foodpackaging equipment. As the peroxycarboxylic acid-containing formulationmay be prepared using GRAS (generally recognized as safe) or food-gradecomponents (enzyme, enzyme substrate, hydrogen peroxide, and buffer),the enzyme-generated peroxycarboxylic acid may also be used fordecontamination of animal carcasses, meat, fruits and vegetables, or fordecontamination of prepared foods. The enzyme-generated peroxycarboxylicacid may be incorporated into a product whose final form is a powder,liquid, gel, film, solid or aerosol. The enzyme-generatedperoxycarboxylic acid may be diluted to a concentration that stillprovides an efficacious decontamination.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can be used to disinfect surfaces and/or objectscontaminated (or suspected of being contaminated) with biologicalcontaminants, such as pathogenic microbial contaminants, by contactingthe surface or object with the products produced by the presentprocesses. As used herein, “contacting” refers to placing a disinfectingcomposition comprising an effective concentration of peroxycarboxylicacid in contact with the surface or inanimate object suspected ofcontamination with a biological contaminant for a period of timesufficient to clean and disinfect. Contacting includes spraying,treating, immersing, flushing, pouring on or in, mixing, combining,painting, coating, applying, affixing to and otherwise communicating aperoxycarboxylic acid solution or composition comprising an efficaciousconcentration of peroxycarboxylic acid, or a solution or compositionthat forms an efficacious concentration of peroxycarboxylic acid, withthe surface or inanimate object suspected of being contaminated with aconcentration of a biological contaminant. The disinfectant compositionsmay be combined with a cleaning composition to provide both cleaning anddisinfection. Alternatively, a cleaning agent (e.g., a surfactant ordetergent) may be incorporated into the formulation to provide bothcleaning and disinfection in a single composition.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can also contain at least one additionalantimicrobial agent, combinations of prion-degrading proteases, avirucide, a sporicide, or a biocide. Combinations of these agents withthe peroxycarboxylic acid produced by the claimed processes can providefor increased and/or synergistic effects when used to clean anddisinfect surfaces and/or objects contaminated (or suspected of beingcontaminated) with biological contaminants. Suitable antimicrobialagents include carboxylic esters (e.g., p-hydroxy alkyl benzoates andalkyl cinnamates); sulfonic acids (e.g., dodecylbenzene sulfonic acid);iodo-compounds or active halogen compounds (e.g., elemental halogens,halogen oxides (e.g., NaOCl, HOCl, HOBr, ClO₂), iodine, interhalides(e.g., iodine monochloride, iodine dichloride, iodine trichloride,iodine tetrachloride, bromine chloride, iodine monobromide, or iodinedibromide), polyhalides, hypochlorite salts, hypochlorous acid,hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins,chlorine dioxide, and sodium chlorite); organic peroxides includingbenzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygengenerators, and mixtures thereof; phenolic derivatives (e.g., o-phenylphenol, o-benzyl-p-chlorophenol, tert-amyl phenol and C₁-C₆ alkylhydroxy benzoates); quaternary ammonium compounds (e.g.,alkyldimethylbenzyl ammonium chloride, dialkyldimethyl ammonium chlorideand mixtures thereof); and mixtures of such antimicrobial agents, in anamount sufficient to provide the desired degree of microbial protection.Effective amounts of antimicrobial agents include about 0.001 wt % toabout 60 wt % antimicrobial agent, about 0.01 wt % to about 15 wt %antimicrobial agent, or about 0.08 wt % to about 2.5 wt % antimicrobialagent.

In one aspect, the peroxycarboxylic acids formed by the process can beused to reduce the concentration of viable biological contaminants (suchas a microbial population) when applied on and/or at a locus. As usedherein, a “locus” comprises part or all of a target surface suitable fordisinfecting or bleaching. Target surfaces include all surfaces that canpotentially be contaminated with biological contaminants. Non-limitingexamples include equipment surfaces found in the food or beverageindustry (such as tanks, conveyors, floors, drains, coolers, freezers,equipment surfaces, walls, valves, belts, pipes, drains, joints,crevasses, combinations thereof, and the like); building surfaces (suchas walls, floors and windows); non-food-industry related pipes anddrains, including water treatment facilities, pools and spas, andfermentation tanks; hospital or veterinary surfaces (such as walls,floors, beds, equipment (such as endoscopes), clothing worn inhospital/veterinary or other healthcare settings, including clothing,scrubs, shoes, and other hospital or veterinary surfaces); restaurantsurfaces; bathroom surfaces; toilets; clothes and shoes; surfaces ofbarns or stables for livestock, such as poultry, cattle, dairy cows,goats, horses and pigs; hatcheries for poultry or for shrimp; andpharmaceutical or biopharmaceutical surfaces (e.g., pharmaceutical orbiopharmaceutical manufacturing equipment, pharmaceutical orbiopharmaceutical ingredients, pharmaceutical or biopharmaceuticalexcipients). Additional hard surfaces include food products, such asbeef, poultry, pork, vegetables, fruits, seafood, combinations thereof,and the like. The locus can also include water absorbent materials suchas infected linens or other textiles. The locus also includes harvestedplants or plant products including seeds, corms, tubers, fruit, andvegetables, growing plants, and especially crop growing plants,including cereals, leaf vegetables and salad crops, root vegetables,legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, wood pulp, paper, vinyl, linoleum, and carpet.

The peroxycarboxylic acids formed by the present process may be used toprovide a benefit to an article of clothing or a textile including, butnot limited to, disinfecting, sanitizing, bleaching, destaining, anddeodorizing. The peroxycarboxylic acids formed by the present processmay be used in any number of laundry care products including, but notlimited to, textile pre-wash treatments, laundry detergents, laundrydetergents or additives, stain removers, bleaching compositions,deodorizing compositions, and rinsing agents, to name a few.

The peroxycarboxylic acids formed by the present process can be used inone or more steps of the wood pulp or paper pulpbleaching/delignification process, particularly where peracetic acid isused (for example, see EP1040222 B1 and U.S. Pat. No. 5,552,018 toDevenyns, J.).

Personal Care Applications

The perhydrolytic enzymes described herein can be used to produce aperacid benefit agent for personal applications such as hair care(bleaching, depilatory), skincare (skin lightening, antimicrobial), andoral care applications (teeth whitening/bleaching or antiseptic), toname a few. The compositions and methods described herein may furthercomprise one or more dermatologically or cosmetically acceptablecomponents known or otherwise effective for use in hair care, skin care,nail care or other personal care products, provided that the optionalcomponents are physically and chemically compatible with the essentialcomponents described herein, or do not otherwise unduly impair productstability, aesthetics, or performance. Non-limiting examples of suchoptional components are disclosed in International Cosmetic IngredientDictionary, Ninth Edition, 2002, and CTFA Cosmetic Ingredient Handbook,Tenth Edition, 2004.

In one embodiment, the dermatologically/cosmetically acceptable carriermay comprise from about 10 wt % to about 99.9 wt %, alternatively fromabout 50 wt % to about 95 wt %, and alternatively from about 75 wt % toabout 95 wt %, of a dermatologically acceptable carrier. Carrierssuitable for use with the composition(s) may include, for example, thoseused in the formulation of hair sprays, mousses, tonics, gels, skinmoisturizers, lotions, and leave-on conditioners. The carrier maycomprise water; organic oils; silicones such as volatile silicones,amino or non-amino silicone gums or oils, and mixtures thereof; mineraloils; plant oils such as olive oil, castor oil, rapeseed oil, coconutoil, wheatgerm oil, sweet almond oil, avocado oil, macadamia oil,apricot oil, safflower oil, candlenut oil, false flax oil, tamanu oil,lemon oil and mixtures thereof; waxes; and organic compounds such asC₂-C₁₀ alkanes, acetone, methyl ethyl ketone, volatile organic C₁-C₁₂alcohols, esters (with the understanding that the choice of ester(s) maybe dependent on whether or not it may act as a carboxylic acid estersubstrates for the perhydrolases) of C₁-C₂₀ acids and of C₁-C₈ alcoholssuch as methyl acetate, butyl acetate, ethyl acetate, and isopropylmyristate, dimethoxyethane, diethoxyethane, C₁₀-C₃₀ fatty alcohols suchas lauryl alcohol, cetyl alcohol, stearyl alcohol, and behenyl alcohol;C₁₀-C₃₀ fatty acids such as lauric acid and stearic acid; C₁₀-C₃₀ fattyamides such as lauric diethanolamide; C₁₀-C₃₀ fatty alkyl esters such asC₁₀-C₃₀ fatty alkyl benzoates; hydroxypropylcellulose; and mixturesthereof. In one embodiment, the carrier comprises water, fatty alcohols,volatile organic alcohols, and mixtures thereof. The composition(s) ofthe present invention further may comprise from about 0.1% to about 10%,and alternatively from about 0.2% to about 5.0%, of a gelling agent tohelp provide the desired viscosity to the composition(s). Non-limitingexamples of suitable optional gelling agents include crosslinkedcarboxylic acid polymers; unneutralized crosslinked carboxylic acidpolymers; unneutralized modified crosslinked carboxylic acid polymers;crosslinked ethylene/maleic anhydride copolymers; unneutralizedcrosslinked ethylene/maleic anhydride copolymers (e.g., EMA 81commercially available from Monsanto); unneutralized crosslinked alkylether/acrylate copolymers (e.g., SALCARE™ SC90 commercially availablefrom Allied Colloids); unneutralized crosslinked copolymers of sodiumpolyacrylate, mineral oil, and PEG-1 trideceth-6 (e.g., SALCARE™ SC91commercially available from Allied Colloids); unneutralized crosslinkedcopolymers of methyl vinyl ether and maleic anhydride (e.g., STABILEZE™QM-PVM/MA copolymer commercially available from International SpecialtyProducts); hydrophobically modified nonionic cellulose polymers;hydrophobically modified ethoxylate urethane polymers (e.g., UCARE™Polyphobe Series of alkali swellable polymers commercially availablefrom Union Carbide); and combinations thereof. In this context, the term“unneutralized” means that the optional polymer and copolymer gellingagent materials contain unneutralized acid monomers. Preferred gellingagents include water-soluble unneutralized crosslinked ethylene/maleicanhydride copolymers, water-soluble unneutralized crosslinked carboxylicacid polymers, water-soluble hydrophobically modified nonionic cellulosepolymers and surfactant/fatty alcohol gel networks such as thosesuitable for use in hair conditioning products.

Recombinant Microbial Expression

The genes and gene products of the instant sequences may be produced inheterologous host cells, particularly in the cells of microbial hosts.Preferred heterologous host cells for expression of the instant genesand nucleic acid molecules are microbial hosts that can be found withinthe fungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi maysuitably host the expression of the present nucleic acid molecules. Theperhydrolase may be expressed intracellularly, extracellularly, or acombination of both intracellularly and extracellularly, whereextracellular expression renders recovery of the desired protein from afermentation product more facile than methods for recovery of proteinproduced by intracellular expression. Transcription, translation and theprotein biosynthetic apparatus remain invariant relative to the cellularfeedstock used to generate cellular biomass; functional genes will beexpressed regardless. Examples of host strains include, but are notlimited to, bacterial, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida,Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas,Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium,Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium,Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia,Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylomicrobium, Methylocystis,Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus,Methanobacterium, Klebsiella, and Myxococcus. In one embodiment,bacterial host strains include Escherichia, Bacillus, and Pseudomonas.In a preferred embodiment, the bacterial host cell is Bacillus subtilisor Escherichia coli.

Industrial Production

A variety of culture methodologies may be applied to produce theperhydrolase catalyst. Large-scale production of a specific gene productover expressed from a recombinant microbial host may be produced bybatch, fed-batch or continuous culture methodologies. Batch andfed-batch culturing methods are common and well known in the art andexamples may be found in Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989) and Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992).

In one embodiment, commercial production of the desired perhydrolasecatalyst is accomplished with a continuous culture. Continuous culturesare an open system where a defined culture media is added continuouslyto a bioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Recovery of the desired perhydrolase catalysts from a batch or fed-batchfermentation, or continuous culture may be accomplished by any of themethods that are known to those skilled in the art. For example, whenthe enzyme catalyst is produced intracellularly, the cell paste isseparated from the culture medium by centrifugation or membranefiltration, optionally washed with water or an aqueous buffer at adesired pH, then a suspension of the cell paste in an aqueous buffer ata desired pH is homogenized to produce a cell extract containing thedesired enzyme catalyst. The cell extract may optionally be filteredthrough an appropriate filter aid such as celite or silica to removecell debris prior to a heat-treatment step to precipitate undesiredprotein from the enzyme catalyst solution. The solution containing thedesired enzyme catalyst may then be separated from the precipitated celldebris and protein produced during the heat-treatment step by membranefiltration or centrifugation, and the resulting partially-purifiedenzyme catalyst solution concentrated by additional membrane filtration,then optionally mixed with an appropriate excipient (for example,maltodextrin, trehalose, sucrose, lactose, sorbitol, mannitol, phosphatebuffer, citrate buffer, or mixtures thereof) and spray-dried to producea solid powder comprising the desired enzyme catalyst. Alternatively,the resulting partially-purified enzyme catalyst solution prepared asdescribed above may be optionally concentrated by additional membranefiltration, and the partially-purified enzyme catalyst solution orresulting enzyme concentrate is then optionally mixed with one or morestabilizing agents (e.g., glycerol, sorbitol, propylene glycol,1,3-propanediol, polyols, polymeric polyols, polyvinylalcohol ormixtures thereof), one or more salts (e.g., sodium chloride, sodiumsulfate, potassium chloride, potassium sulfate, or mixtures thereof),and one or more biocides, and maintained as an aqueous solution untilused.

When an amount, concentration, or other value or parameter is giveneither as a range, preferred range, or a list of upper preferable valuesand lower preferable values, this is to be understood as specificallydisclosing all ranges formed from any pair of any upper range limit orpreferred value and any lower range limit or preferred value, regardlessof whether ranges are separately disclosed. Where a range of numericalvalues is recited herein, unless otherwise stated, the range is intendedto include the endpoints thereof, and all integers and fractions withinthe range. It is not intended that the scope be limited to the specificvalues recited when defining a range.

General Methods

The following examples are provided to demonstrate differentembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the methods disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the presently disclosed methods.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.) orSigma-Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “L”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “ppm”means part(s) per million, “wt” means weight, “wt %” means weightpercent, “g” means gram(s), “g” means microgram(s), “ng” meansnanogram(s), “g” means gravity, “HPLC” means high performance liquidchromatography, “dd H₂O” means distilled and deionized water, “dcw”means dry cell weight, “ATCC” or “ATCC®” means the American Type CultureCollection (Manassas, Va.), “U” means unit(s) of perhydrolase activity,“rpm” means revolution(s) per minute, “EDTA” meansethylenediaminetetraacetic acid, “IPTG” meansisopropyl-13-D-thio-galactoside, “BCA” means bicinchoninic acid, and“ABTS” means 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate).

Example 1 Cloning and Production of CE-7 Acetyl Xylan Esterase fromActinosynnema mirum in E. coli

The gene encoding the acetyl xylan esterase enzyme from Actinosynnemamirum as reported in GENBANK® (Accession No. ACU35776.1; GI:255920265)was synthesized using codons optimized for expression in E. coli (DNA2.0, Menlo Park, Calif.). The nucleic acid product (SEQ ID NO: 3) wassubcloned into PJEXPRESS404® (DNA 2.0, Menlo Park, Calif.) to generatethe plasmid identified as pMP91. The plasmid pMP91 was used to transformE. coli KLP18 (described in U.S. Pat. No. 7,723,083) to generate thestrain identified as KLP18/pMP91. KLP18/pMP91 was grown in LB media at37° C. with shaking up to OD_(600nm)=0.4-0.5, at which time IPTG wasadded to a final concentration of 1 mM, and incubation continued for 2-3h. Cells were harvested by centrifugation at 5,000×g for 15 minutes thenresuspended (20% w/v) in 50 mM potassium phosphate buffer pH 7.0supplemented with 1.0 mM dithiothreitol. Resuspended cells were passedthrough a French pressure cell twice. Lysed cells were centrifuged for30 minutes at 12,000×g and the protein concentration in the extractsupernatant was determined using a BCA assay kit (Sigma-Aldrich, St.Louis, Mo.). SDS-PAGE was used to confirm expression of the CE-7 enzyme(SEQ ID NO: 4), and analysis of the gels using ImageJ, a public domainJava image processing program, indicated that perhydrolase constituted11% of total soluble protein.

Example 2 Cloning and Production of CE-7 Acetyl Xylan Esterase fromPropionibacterium acnes in E. coli

The gene encoding the acetyl xylan esterase enzyme fromPropionibacterium acnes as reported in GENBANK® (Accession no.AEE71478.1; GI:332674662) was synthesized using codons optimized forexpression in E. coli (DNA 2.0, Menlo Park, Calif.). The nucleic acidproduct (SEQ ID NO: 5) was subcloned into PJEXPRESS404® (DNA 2.0, MenloPark, Calif.) to generate the plasmid identified as pMP92. The plasmidpMP92 was used to transform E. coli KLP18 (described in U.S. Pat. No.7,723,083) to generate the strain identified as KLP18/pMP92. KLP18/pMP92was grown in LB media at 37° C. with shaking up to OD_(600nm)=0.4-0.5,at which time IPTG was added to a final concentration of 1 mM, andincubation continued for 2-3 h. Cells were harvested by centrifugationat 5,000×g for 15 minutes then resuspended (20% w/v) in 50 mM potassiumphosphate buffer pH 7.0 supplemented with 1.0 mM dithiothreitol.Resuspended cells were passed through a French pressure cell twice.Lysed cells were centrifuged for 30 minutes at 12,000×g and the totalsoluble protein concentration in the extract supernatant was determinedusing a BCA assay kit (Sigma-Aldrich, St. Louis, Mo.). SDS-PAGE was usedto confirm expression of the CE-7 enzyme (SEQ ID NO: 6), and analysis ofthe gels using ImageJ, a public domain Java image processing program,indicated that perhydrolase constituted 13% of total soluble protein.

Example 3

Cloning and Production of CE-7 Acetyl Xylan Esterase from Streptococcusecui in E. coli

The gene encoding the acetyl xylan esterase enzyme from Streptococcusequi as reported in GENBANK® (Accession no. CAX00506.1: GI: 225702544)was synthesized using codons optimized for expression in E. coli (DNA2.0, Menlo Park, Calif.). The nucleic acid product (SEQ ID NO: 7) wassubcloned into PJEXPRESS404® (DNA 2.0, Menlo Park, Calif.) to generatethe plasmid identified as pMP93. The plasmid pMP93 was used to transformE. coli KLP18 (described in U.S. Pat. No. 7,723,083) to generate thestrain identified as KLP18/pMP93.

KLP18/pMP93 was grown in LB media at 37° C. with shaking up toOD_(600nm)=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h. Cells wereharvested by centrifugation at 5,000×g for 15 minutes then resuspended(20% w/v) in 50 mM potassium phosphate buffer pH 7.0 supplemented with1.0 mM dithiothreitol. Resuspended cells were passed through a Frenchpressure cell twice. Lysed cells were centrifuged for 30 minutes at12,000×g and the total soluble protein concentration in the extractsupernatant was determined using a BCA assay kit (Sigma-Aldrich, St.Louis, Mo.). SDS-PAGE was used to confirm expression of the CE-7 enzyme(SEQ ID NO: 8), and analysis of the gels using ImageJ, a public domainJava image processing program, indicated that perhydrolase constituted26% of total soluble protein.

Example 4 Cloning and Production of CE-7 Acetyl Xylan Esterase fromStackebrandtia nassauensis in E. coli

The gene encoding the acetyl xylan esterase enzyme from Stackebrandtianassauensis as reported in GENBANK® (Accession No. ADD42786.1:GI:290569821) was synthesized using codons optimized for expression inE. coli (DNA 2.0, Menlo Park, Calif.). The nucleic acid product (SEQ IDNO: 9) was subcloned into PJEXPRESS404® (DNA 2.0, Menlo Park, Calif.) togenerate the plasmid identified as pMP94. The plasmid pMP91 was used totransform E. coli KLP18 (described in U.S. Pat. No. 7,723,083) togenerate the strain identified as KLP18/pMP94. KLP18/pMP94 was grown inLB media at 37° C. with shaking up to OD_(600nm)=0.4-0.5, at which timeIPTG was added to a final concentration of 1 mM, and incubationcontinued for 2-3 h. Cells were harvested by centrifugation at 5,000×gfor 15 minutes then resuspended (20% w/v) in 50 mM potassium phosphatebuffer pH 7.0 supplemented with 1.0 mM dithiothreitol. Resuspended cellswere passed through a French pressure cell twice. Lysed cells werecentrifuged for 30 minutes at 12,000×g and the total soluble proteinconcentration in the extract supernatant was determined using a BCAassay kit (Sigma-Aldrich, St. Louis, Mo.). SDS-PAGE was used to confirmexpression of the CE-7 enzyme (SEQ ID NO: 10), and analysis of the gelsusing ImageJ, a public domain Java image processing program, indicatedthat perhydrolase constituted 33% of total soluble protein.

Example 5 Cloning and Production of CE-7 Acetyl Xylan Esterase fromStreptococcus agalactiae in E. coli

The gene encoding the acetyl xylan esterase enzyme from Streptococcusagalactiae as reported in GENBANK® (Accession No. AAM98949.1;GI:22533045) was synthesized using codons optimized for expression in E.coli (DNA 2.0, Menlo Park, Calif.). The nucleic acid product (SEQ ID NO:11) was subcloned into PJEXPRESS404® (DNA 2.0, Menlo Park, Calif.) togenerate the plasmid identified as pMP95. The plasmid pMP95 was used totransform E. coli KLP18 (described in U.S. Pat. No. 7,723,083) togenerate the strain identified as KLP18/pMP95. KLP18/pMP95 was grown inLB media at 37° C. with shaking up to OD_(600nm)=0.4-0.5, at which timeIPTG was added to a final concentration of 1 mM, and incubationcontinued for 2-3 h. Cells were harvested by centrifugation at 5,000×gfor 15 minutes then resuspended (20% w/v) in 50 mM potassium phosphatebuffer pH 7.0 supplemented with 1.0 mM dithiothreitol. Resuspended cellswere passed through a French pressure cell twice. Lysed cells werecentrifuged for 30 minutes at 12,000×g and the total soluble proteinconcentration in the extract supernatant was determined using a BCAassay kit (Sigma-Aldrich, St. Louis, Mo.). SDS-PAGE was used to confirmexpression of the CE-7 enzyme (SEQ ID NO: 12), and analysis of the gelsusing ImageJ, a public domain Java image processing program, indicatedthat perhydrolase constituted 7.3% of total soluble protein.

Example 6 Perhydrolase Activity Assays

Perhydrolase activity in extract supernatant was determined by reactionscontaining 22.5 mM triacetin, 22.5 mM hydrogen peroxide and 6.25 μgextract supernatant total soluble protein/mL. Incubation was for 10minutes at ambient temperature (22-24° C.). Reactions were stopped byadding an equal volume of 1.25 M phosphoric acid containing 100 mMortho-phenylenediamine. After 30 minutes, the absorbance at 458 nm wasmeasured (Table 1). Additional perhydrolase activity measurements weremade in reactions containing 10 mM triacetin and 10 mM hydrogen peroxideor 50 mM triacetin and 50 mM hydrogen peroxide (Table 1). CE-7 acetylxylan esterase from T. maritima was also produced in E. coli KLP18(described in US Patent Application Publication 2008-0176299) and usedas a positive control for the perhydrolase assay. E. coli KLP18 extractnot containing a CE-7 enzyme was used a negative control.

TABLE 1 OD 458 nm triacetin 10 mM 22.5 mM 50 mM CE-7 esterase H₂O₂source ref SEQ ID NO: 10 mM 22.5 mM 50 mM A. mirum Ami 4 0.6 2.3 2.9 P.acnes Pac 6 0.4 1.2 2.5 S. equi Seq 8 0.3 1.9 2.0 S. nassauensis Sna 100.1 0.2 0.8 S. agalactiae Sag 12 0.0 0.1 0.3 T. maritima Tma 2 0.2 1.02.5 none (control) 0.0 0.0 0.0

Example 7 Production of Peracetic Acid from Triacetin and HydrogenPeroxide by CE-7 Esterases

Reactions (10 mL total volume) were run at 25° C. in potassium phosphatebuffer (50 mM, pH 7.0) containing triacetin (10 mM), hydrogen peroxide(20 mM) and 5.0 μg/mL of extract supernatant total soluble proteincontaining the CE-7 esterase from Actinosynnema mirum (SEQ ID NO: 4),Propionibacterium acnes (SEQ ID NO: 6), Streptococcus equi (SEQ ID NO:8), Stackebrandtia nassauensis (SEQ ID NO: 10) or Streptococcusagalactiae (SEQ ID NO: 12), prepared as described in Examples 1-5.Reactions were stirred for only the first 45 seconds of reaction toinitially mix the reactants and enzyme. A comparative control reactionwas run under identical conditions to that described immediately aboveusing 5.0 μg/mL of extract total soluble protein isolated from E. coliKLP18 (used to express the CE-7 esterases), where the extractsupernatant was prepared according to the procedure of Example 1. Asecond comparative control reaction was also run under identicalconditions to that described immediately above using no added extractsupernatant total soluble protein, where peracetic acid produced in theabsence of added esterase was the result of chemical perhydrolysis oftriacetin by hydrogen peroxide under the specified reaction conditions.The CE-7 acetyl xylan esterase from T. maritima (SEQ ID NO: 2) was alsoproduced in E. coli KLP18 (described in US Patent ApplicationPublication 2008-0176299) and used as a positive control in acomparative reaction (11% of total soluble protein in cell extractsupernatant).

Analysis of reaction samples for the production of peracetic acidfollowed the method described in Pinkernell et. al. (Analyst, 122:567(1997)) using colorimetric detection of ABTS oxidation by peraceticacid. A 50 μL reaction sample was added to 950 μL of 5 mM H₃PO₄ to stopthe enzymatic reaction (final pH between pH 2-3), and 50 μL of theresulting solution was added to a 96-well microtiter plate wellcontaining 200 μL of an aqueous solution containing 0.25 M acetic acid,0.125 g/L ABTS and 5.0 mg/L of KI. The solution was allowed to developfor 5 min, then the absorbance of the solution was measured at 405 nmusing a microplate reader. The peracetic acid concentration in eachsample was calculated from a standard curve developed simultaneouslyusing a peracetic acid reagent solution (Table 2).

TABLE 2 CE-7 perhydrolase-catalyzed production of peracetic acid (PAA)from triacetin and hydrogen peroxide in potassium phosphate buffer (50mM, pH 7.0) at 25° C. total soluble PAA at PAA at PAA at PAA at PAA atSEQ ID triacetin H₂O₂ protein 2 min 5 min 10 min 20 min 30 min CE-7esterase source NO (mM) (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm)control - no enzyme 10 20 0 4.1 4.3 3.1 4.0 4.6 control - E. coli KLP1810 20 5.0 3.9 4.0 4.1 4.0 4.8 A. mirum 4 10 20 5.0 8.8 17 30 50 68 P.acnes 6 10 20 5.0 6.4 12 18 28 36 S. equi 8 10 20 5.0 18 33 47 53 52 S.nassauensis 10 10 20 5.0 4.3 6.0 7.8 11 15 S. agalactiae 12 10 20 5.04.3 4.5 5.1 6.6 7.4 T. maritima 2 10 20 5.0 5.6 11 16 28 43

Example 8 Production of Peracetic Acid from Propylene Glycol Diacetateand Hydrogen Peroxide by CE-7 Esterases

Reactions (10 mL total volume) were run at 25° C. in potassium phosphatebuffer (50 mM, pH 7.0) containing propylene glycol diacetate (10 mM),hydrogen peroxide (20 mM) and 5.0 μg/mL of extract supernatant totalsoluble protein containing the CE-7 esterase from Actinosynnema mirum(SEQ ID NO: 4), Propionibacterium acnes (SEQ ID NO: 6), Streptococcusequi (SEQ ID NO: 8), or Stackebrandtia nassauensis (SEQ ID NO: 10)prepared as described in Examples 1-4. Reactions were stirred for onlythe first 45 seconds of reaction to initially mix the reactants andenzyme. A comparative control reaction was run under identicalconditions to that described immediately above using 5.0 μg/mL ofextract total soluble protein isolated from E. coli KLP18 (used toexpress the CE-7 esterases), where the extract supernatant was preparedaccording to the procedure of Example 1. A second comparative controlreaction was also run under identical conditions to that describedimmediately above using no added extract supernatant total solubleprotein, where peracetic acid produced in the absence of added esterasewas the result of chemical perhydrolysis of propylene glycol diacetateby hydrogen peroxide under the specified reaction conditions. The CE-7acetyl xylan esterase from T. maritima (SEQ ID NO: 2) was also producedin E. coli KLP18 (described in US Patent Application Publication2008-0176299) and used as a positive control in a comparative reaction(11% of total soluble protein in cell extract supernatant). Analysis ofreaction samples for the production of peracetic acid followed themethod described in Example 7 (Table 3).

TABLE 3 CE-7 perhydrolase-catalyzed production of peracetic acid (PAA)from propylene glycol diacetate and hydrogen peroxide in potassiumphosphate buffer (50 mM, pH 7.0) at 25° C. propylene total soluble PAAat PAA at PAA at PAA at PAA at SEQ ID glycol diacetate H₂O₂ protein 2min 5 min 10 min 20 min 30 min CE-7 esterase source NO (mM) (mM) (μg/mL)(ppm) (ppm) (ppm) (ppm) (ppm) control - no enzyme 10 20 0 5.4 5.0 5.05.2 5.1 control - E. coli KLP18 10 20 5.0 5.4 5.1 5.2 5.1 5.2 A. mirum 410 20 5.0 8.0 13 22 35 47 P. acnes 6 10 20 5.0 6.1 6.9 8.6 13 12 S. equi8 10 20 5.0 6.6 11 11 12 12 S. nassauensis 10 10 20 5.0 4.6 4.8 8.2 6.47.6 T. maritima 2 10 20 5.0 5.7 6.8 8.3 12 15

Example 9 Production of Peracetic Acid from α-D-Glucose Pentaacetate andHydrogen Peroxide by CE-7 Esterases

Reactions (10 mL total volume) were run at 25° C. in potassium phosphatebuffer (50 mM, pH 7.0) containing α-D-glucose pentaacetate (10 mM),hydrogen peroxide (20 mM) and 5.0 μg/mL of extract supernatant totalsoluble protein containing the CE-7 esterase from Actinosynnema mirum(SEQ ID NO: 4), Streptococcus equi (SEQ ID NO: 8), or Streptococcusagalactiae (SEQ ID NO: 12) prepared as described in Examples 1, 3 and 5.Reactions were stirred for only the first 45 seconds of reaction toinitially mix the reactants and enzyme. A comparative control reactionwas run under identical conditions to that described immediately aboveusing 5.0 μg/mL of extract total soluble protein isolated from E. coliKLP18 (used to express the CE-7 esterases), where the extractsupernatant was prepared according to the procedure of Example 1. Asecond comparative control reaction was also run under identicalconditions to that described immediately above using no added extractsupernatant total soluble protein, where peracetic acid produced in theabsence of added esterase was the result of chemical perhydrolysis ofα-D-glucose pentaacetate by hydrogen peroxide under the specifiedreaction conditions. The CE-7 acetyl xylan esterase from T. maritima(SEQ ID NO: 2) was also produced in E. coli KLP18 (described in USPatent Application Publication 2008-0176299) and used as a positivecontrol in a comparative reaction (11% of total soluble protein in cellextract supernatant). Analysis of reaction samples for the production ofperacetic acid followed the method described in Example 7 (Table 4).

TABLE 4 CE-7 perhydrolase-catalyzed production of peracetic acid (PAA)from α-D-glucose pentaacetate and hydrogen peroxide in potassiumphosphate buffer (50 mM, pH 7.0) at 25° C. α-D-glucose total soluble PAAat PAA at PAA at PAA at PAA at SEQ ID pentaacetate H₂O₂ protein 2 min 5min 10 min 20 min 30 min CE-7 esterase source NO (mM) (mM) (μg/mL) (ppm)(ppm) (ppm) (ppm) (ppm) control - no enzyme 10 20 0 6.7 18 41 93 136control - E. coli KLP18 10 20 5.0 8.7 20 46 94 137 A. mirum 4 10 20 5.020 34 68 130 179 S. equi 8 10 20 5.0 11 23 46 93 141 S. agalactiae 12 1020 5.0 16 29 51 99 148 T. maritima 2 10 20 5.0 11 24 47 99 144

Example 10 Production of Peracetic Acid from D-Sorbitol Hexaacetate andHydrogen Peroxide by CE-7 Esterases

Reactions (10 mL total volume) were run at 25° C. in potassium phosphatebuffer (50 mM, pH 7.0) containing D-sorbitol hexaacetate (10 mM),hydrogen peroxide (20 mM) and 5.0 μg/mL of extract supernatant totalsoluble protein containing the CE-7 esterase from Actinosynnema mirum(SEQ ID NO: 4), or Streptococcus equi (SEQ ID NO: 8) prepared asdescribed in Examples 1 and 3. Reactions were stirred for only the first45 seconds of reaction to initially mix the reactants and enzyme. Acomparative control reaction was run under identical conditions to thatdescribed immediately above using 5.0 μg/mL of extract total solubleprotein isolated from E. coli KLP18 (used to express the CE-7esterases), where the extract supernatant was prepared according to theprocedure of Example 1. A second comparative control reaction was alsorun under identical conditions to that described immediately above usingno added extract supernatant total soluble protein, where peracetic acidproduced in the absence of added esterase was the result of chemicalperhydrolysis of D-sorbitol hexaacetate by hydrogen peroxide under thespecified reaction conditions. The CE-7 acetyl xylan esterase from T.maritima (SEQ ID NO: 2) was also produced in E. coli KLP18 (described inUS Patent Application Publication 2008-0176299) and used as a positivecontrol in a comparative reaction (11% of total soluble protein in cellextract supernatant). Analysis of reaction samples for the production ofperacetic acid followed the method described in Example 7 (Table 5).

TABLE 5 CE-7 perhydrolase-catalyzed production of peracetic acid (PAA)from D-sorbitol hexaacetate and hydrogen peroxide in potassium phosphatebuffer (50 mM, pH 7.0) at 25° C. D-sorbitol total soluble PAA at PAA atPAA at PAA at PAA at SEQ ID hexaacetate H₂O₂ protein 2 min 5 min 10 min20 min 30 min CE-7 esterase source NO (mM) (mM) (μg/mL) (ppm) (ppm)(ppm) (ppm) (ppm) control - no enzyme 10 20 0 3.6 3.9 4.4 4.2 4.5control - E. coli KLP18 10 20 5.0 3.8 4.2 4.1 4.1 4.7 A. mirum 4 10 205.0 5.6 9.0 15 25 34 S. equi 8 10 20 5.0 4.8 5.9 7.7 9.1 9.8 T. maritima2 10 20 5.0 5.0 6.0 11 15 21

Example 11 Production of Peracetic Acid from Tri-O-Acetyl-D-Glucal andHydrogen Peroxide by CE-7 Esterases

Reactions (50 mL total volume) were run at 25° C. in potassium phosphatebuffer (10 mM, pH 7.0) containing tri-O-acetyl-D-glucal (2 mM), hydrogenperoxide (10 mM) and 5.0 μg/mL of extract supernatant total solubleprotein containing the CE-7 esterase from Actinosynnema mirum (SEQ IDNO: 4) or Streptococcus equi (SEQ ID NO: 8) prepared as described inExamples 1 and 3. Reactions were stirred for only the first 45 secondsof reaction to initially mix the reactants and enzyme. A comparativecontrol reaction was run under identical conditions to that describedimmediately above using 5.0 μg/mL of extract total soluble proteinisolated from E. coli KLP18 (used to express the CE-7 esterases), wherethe extract supernatant was prepared according to the procedure ofExample 1. A second comparative control reaction was also run underidentical conditions to that described immediately above using no addedextract supernatant total soluble protein, where peracetic acid producedin the absence of added esterase was the result of chemicalperhydrolysis of tri-O-acetyl-D-glucal by hydrogen peroxide under thespecified reaction conditions. The CE-7 acetyl xylan esterase from T.maritima (SEQ ID NO: 2) was also produced in E. coli KLP18 (described inUS Patent Application Publication 2008-0176299) and used as a positivecontrol in a comparative reaction (11% of total soluble protein in cellextract supernatant). Analysis of reaction samples for the production ofperacetic acid followed the method described in Example 7 (Table 6).

TABLE 6 CE-7 perhydrolase-catalyzed production of peracetic acid (PAA)from tri-O-acetyl-D-glucal and hydrogen peroxide in potassium phosphatebuffer (10 mM, pH 7.0) at 25° C. tri-O-acetyl-D- total soluble PAA atPAA at SEQ ID glucal H₂O₂ protein 5 min 20 min CE-7 esterase source NO:(mM) (mM) (μg/mL) (ppm) (ppm) control - no enzyme 2 10 0 2.6 2.2control - E. coli KLP18 2 10 5.0 2.5 1.5 A. mirum 4 2 10 5.0 6.7 18 S.equi 8 2 10 5.0 5.8 6.4 T. maritima 2 2 10 5.0 4.9 13

Example 12 Production of Peracetic Acid from 4-(Acetyloxy)-Benzoic Acidand Hydrogen Peroxide by CE-7 Esterases

Reactions (10 mL total volume) were run at 20° C. in potassium phosphatebuffer (50 mM, pH 7.0) containing 4-(acetyloxy)-benzoic acid (CAS2345-34-8; 25 mM), hydrogen peroxide (20 mM) and 5.0 μg/mL of extractsupernatant total soluble protein containing the CE-7 esterase fromActinosynnema mirum (SEQ ID NO: 4), Propionibacterium acnes (SEQ ID NO:6), Streptococcus equi (SEQ ID NO: 8), or Stackebrandtia nassauensis(SEQ ID NO: 10) prepared as described in Examples 1-4. Reactions werestirred for only the first 45 seconds of reaction to initially mix thereactants and enzyme. A comparative control reaction was run underidentical conditions to that described immediately above using 5.0 μg/mLof heat-treated extract total soluble protein isolated from E. coliKLP18 (used to express the CE-7 esterases), where the extractsupernatant was prepared according to the procedure of Example 1. Asecond comparative control reaction was also run under identicalconditions to that described immediately above using no added extractsupernatant total soluble protein, where peracetic acid produced in theabsence of added esterase was the result of chemical perhydrolysis of4-(acetyloxy)-benzoic acid by hydrogen peroxide under the specifiedreaction conditions. The CE-7 acetyl xylan esterase from T. maritima(SEQ ID NO: 2) was also produced in E. coli KLP18 (described in USPatent Application Publication 2008-0176299) and used as a positivecontrol in a comparative reaction (11% of total soluble protein in cellextract supernatant). Analysis of reaction samples for the production ofperacetic acid followed the method described in Example 7 (Table 7).

TABLE 7 CE-7 perhydrolase-catalyzed production of peracetic acid (PAA)from 4-(acetyloxy)-benzoic acid and hydrogen peroxide in potassiumphosphate buffer (50 mM, pH 7.0) at 20° C. 4-(acetyloxy)- total solublePAA at PAA at SEQ ID benzoic acid H₂O₂ protein 5 min 20 min CE-7esterase source NO (mM) (mM) (μg/mL) (ppm) (ppm) control - no enzyme 2520 0 28 74 control - E. coli KLP18 25 20 5.0 21 73 A. mirum 4 25 20 5.036 85 P. acnes 6 25 20 5.0 31 90 S. equi 8 25 20 5.0 31 98 S.nassauensis 10 25 20 5.0 29 80 T. maritima 2 25 20 5.0 21 71

Example 13 Cloning and Production of an Actinosynnema mirum Acetyl XylanEsterase Variant

A gene encoding a variant of the acetyl xylan esterase enzyme from A.mirum as reported in GENBANK® (Accession No. ACU35776.1: GI: 255920265)was synthesized using codons optimized for expression in E. coli (DNA2.0, Menlo Park, Calif.). The nucleic acid product (SEQ ID NO: 13) wassubcloned into PJEXPRESS404® (DNA 2.0, Menlo Park, Calif.) to generatethe plasmid identified as pMP91a. The encoded variant protein is calledAmi_C276S (SEQ ID NO: 14). The plasmid pMP91a was used to transform E.coli KLP18 (described in U.S. Pat. No. 7,723,083) to generate the strainidentified as KLP18/pMP91a.

KLP18/pMP91a was grown in LB media at 37° C. with shaking up toOD_(600nm)=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h. Cells wereharvested by centrifugation at 5,000×g for 15 minutes then resuspended(20% w/v) in 50 mM potassium phosphate buffer pH 7.0 supplemented with1.0 mM dithiothreitol. Resuspended cells were passed through a Frenchpressure cell twice. Lysed cells were centrifuged for 30 minutes at12,000×g and the protein concentration in the supernatant was determinedusing a BCA assay kit (Sigma-Aldrich, St. Louis, Mo.). SDS-PAGE was usedto confirm expression of the enzyme and densitometry (ImageJ software,National Institutes of Health, Bethesda, Md.) was used to calculate theenzyme protein as approximately 16-18% of the total protein.

Example 14 Cloning and Production of an Actinosynnema mirum Acetyl XylanEsterase Variant

A gene encoding a variant of the acetyl xylan esterase enzyme from A.mirum as reported in GENBANK® (Accession No. ACU35776.1: GI: 255920265)was synthesized using codons optimized for expression in E. coli (DNA2.0, Menlo Park, Calif.). The nucleic acid product (SEQ ID NO: 15) wassubcloned into PJEXPRESS404® (DNA 2.0, Menlo Park, Calif.) to generatethe plasmid identified as pMP91b. The encoded variant protein is calledAmi_C276T (SEQ ID NO: 16). The plasmid pMP91b was used to transform E.coli KLP18 (described in U.S. Pat. No. 7,723,083) to generate the strainidentified as KLP18/pMP91b. KLP18/pMP91b was grown in LB media at 37° C.with shaking up to OD_(600nm)=0.4-0.5, at which time IPTG was added to afinal concentration of 1 mM, and incubation continued for 2-3 h. Cellswere harvested by centrifugation at 5,000×g for 15 minutes thenresuspended (20% w/v) in 50 mM potassium phosphate buffer pH 7.0supplemented with 1.0 mM dithiothreitol. Resuspended cells were passedthrough a French pressure cell twice. Lysed cells were centrifuged for30 minutes at 12,000×g and the protein concentration in the supernatantwas determined using a BCA assay kit (Sigma-Aldrich, St. Louis, Mo.).SDS-PAGE was used to confirm expression of the enzyme and densitometry(ImageJ software, National Institutes of Health, Bethesda, Md.) was usedto calculate the enzyme protein as approximately 16-18% of the totalprotein.

Example 15 Perhydrolase Activity Assays

Perhydrolase activity in extracts was determined by reactions containing22.5 mM triacetin, 22.5 mM hydrogen peroxide and 1.5 μg totalprotein/mL. Incubation was for 10 minutes at ambient temperature (22-24°C.). Reactions were stopped by adding an equal volume of 1.25 Mphosphoric acid containing 100 mM ortho-phenylenediamine. After 30minutes, the absorbance at 458 nm was measured (Table 8). E. coli KLP18extract not containing an acetyl xylan esterase enzyme was used anegative control.

TABLE 8 Enzyme ID. SEQ ID NO: OD 458 nm Ami_wt 4 0.21 Ami_C276S 14 2.3Ami_C276T 16 1.5 Control - no enzyme 0

Example 16 Production of Peracetic Acid from Triacetin and HydrogenPeroxide by CE-7 Esterase Variants

Reactions (10 mL total volume) were run at 25° C. in potassium phosphatebuffer (20 mM, pH 7.0) containing triacetin (0.75 mM), hydrogen peroxide(1.4 mM) and either 1.0 μg/mL or 2.0 μg/mL of CE-7 esterase fromwild-type Actinosynnema mirum (SEQ ID NO: 4, prepared as described inExample 1), Actinosynnema mirum C276S variant (SEQ ID NO: 14, preparedas described in Example 13), Actinosynnema mirum C276T variant (SEQ IDNO: 16, prepared as described in Example 14), T. maritima C277S (SEQ IDNO: 17, produced in E. coli KLP18 as described in U.S. Pat. No.8,062,875), and T. maritima C277T (SEQ ID NO:18, produced in E. coliKLP18 as described in U.S. Pat. No. 8,062,875). Analysis of cellextracts containing CE-7 esterase by SDS-PAGE gels in combination withanalysis of the gels using ImageJ, a public domain Java image processingprogram, was used to calculate the concentration of CE-7 esterase incell extracts as a percentage of total soluble protein. Reactions werestirred for only the first 45 seconds of reaction to initially mix thereactants and enzyme. A comparative control reaction was run underidentical conditions to that described immediately above using no addedCE-7 esterase, where peracetic acid produced in the absence of addedesterase was the result of chemical perhydrolysis of triacetin byhydrogen peroxide under the specified reaction conditions. Analysis ofreaction samples for the production of peracetic acid followed themethod described in Example 7 (Table 9).

TABLE 9 CE-7 perhydrolase variant-catalyzed production of peracetic acid(PAA) from triacetin and hydrogen peroxide in potassium phosphate buffer(20 mM, pH 7.0) at 25° C. CE-7 esterase PAA at PAA at PAA at PAA at PAAat SEQ ID triacetin H₂O₂ variant 2 min 5 min 10 min 20 min 30 min CE-7esterase source NO (mM) (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm)control - no enzyme 0.75 1.4 0 0.5 0.4 0.4 0.4 0.3 A. mirum wild-type 40.75 1.4 1.0 0.6 1.4 2.1 1.2 0.6 A. mirum C276S 14 0.75 1.4 1.0 3.1 5.46.8 4.9 3.0 A. mirum C276T 16 0.75 1.4 1.0 3.0 6.0 8.8 8.1 5.1 T.maritima C277S 17 0.75 1.4 1.0 1.3 2.6 4.2 4.8 4.2 T. maritima C277T 180.75 1.4 1.0 2.4 4.8 6.1 5.6 3.7 A. mirum wild-type 4 0.75 1.4 2.0 1.01.8 1.2 0.4 0.4 A. mirum C276S 14 0.75 1.4 2.0 5.3 6.7 4.8 1.8 1.2 A.mirum C276T 16 0.75 1.4 2.0 5.5 8.6 7.7 2.4 1.4 T. maritima C277S 170.75 1.4 2.0 2.9 4.2 5.0 3.4 2.0 T. maritima C277T 18 0.75 1.4 2.0 4.16.2 5.5 2.1 1.1

Example 17 Production of Peracetic Acid from Triacetin and HydrogenPeroxide by CE-7 Esterase Variants

Reactions (10 mL total volume) were run at 25° C. in potassium phosphatebuffer (50 mM, pH 7.0) containing triacetin (10 mM), hydrogen peroxide(20 mM) and 0.5 μg/mL of CE-7 esterase from wild-type Actinosynnemamirum (SEQ ID NO: 4, prepared as described in Example 1), Actinosynnemamirum C276S variant (SEQ ID NO: 14, prepared as described in Example13), Actinosynnema mirum C276T variant (SEQ ID NO: 16, prepared asdescribed in Example 14), T. maritima C277S (SEQ ID NO: 17), and T.maritima C277T (SEQ ID NO: 18). Analysis of cell extracts containingCE-7 esterase by SDS-PAGE gels in combination with analysis of the gelsusing ImageJ, a public domain Java image processing program, was used tocalculate the concentration of CE-7 esterase in cell extracts as apercentage of total soluble protein. Reactions were stirred for only thefirst 45 seconds of reaction to initially mix the reactants and enzyme.A comparative control reaction was run under identical conditions tothat described immediately above using no added CE-7 esterase, whereperacetic acid produced in the absence of added esterase was the resultof chemical perhydrolysis of triacetin by hydrogen peroxide under thespecified reaction conditions. Analysis of reaction samples for theproduction of peracetic acid followed the method described in Example 7(Table 10).

TABLE 10 CE-7 perhydrolase variant-catalyzed production of peraceticacid (PAA) from triacetin and hydrogen peroxide in potassium phosphatebuffer (50 mM, pH 7.0) at 25° C. CE-7 esterase PAA at PAA at PAA at PAAat PAA at SEQ ID triacetin H₂O₂ variant 2 min 5 min 10 min 20 min 30 minCE-7 esterase source NO (mM) (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm)control - no enzyme 10 20 0 3.1 8.0 2.6 4.1 4.0 A. mirum wild-type 4 1020 0.5 8.1 11 19 34 46 A. mirum C276S 14 10 20 0.5 39 76 99 92 80 A.mirum C276T 16 10 20 0.5 26 60 96 118 115 T. maritima C277S 17 10 20 0.522 43 63 74 68 T. maritima C277T 18 10 20 0.5 29 58 96 131 132

Example 18 Production of Peracetic Acid from Triacetin and HydrogenPeroxide by CE-7 Esterase Variants

Reactions (10 mL total volume) were run at 25° C. in sodium carbonatebuffer (20 mM, pH 10.5) containing triacetin (0.75 mM), hydrogenperoxide (1.4 mM, from sodium percarbonate) and either 1.0 μg/mL or 2.0μg/mL of CE-7 esterase from wild-type Actinosynnema mirum (SEQ ID NO: 4,prepared as described in Example 1), Actinosynnema mirum C276S variant(SEQ ID NO: 14, prepared as described in Example 13), Actinosynnemamirum C276T variant (SEQ ID NO:16, prepared as described in Example 14),T. maritima C277S (SEQ ID NO: 17), and T. maritima C277T (SEQ ID NO:18). Analysis of cell extracts containing CE-7 esterase by SDS-PAGE gelsin combination with analysis of the gels using ImageJ, a public domainJava image processing program, was used to calculate the concentrationof CE-7 esterase in cell extracts as a percentage of total solubleprotein. Reactions were stirred for only the first 45 seconds ofreaction to initially mix the reactants and enzyme. A comparativecontrol reaction was run under identical conditions to that describedimmediately above using no added CE-7 esterase, where peracetic acidproduced in the absence of added esterase was the result of chemicalperhydrolysis of triacetin by hydrogen peroxide under the specifiedreaction conditions. Analysis of reaction samples for the production ofperacetic acid followed the method described in Example 7 (Table 11).

TABLE 11 CE-7 perhydrolase variant-catalyzed production of peraceticacid (PAA) from triacetin and hydrogen peroxide in sodium carbonatebuffer (20 mM, pH 10.5) at 25° C. CE-7 esterase PAA at PAA at PAA at PAAat PAA at SEQ ID triacetin H₂O₂ variant 2 min 5 min 10 min 20 min 30 minCE-7 esterase source NO (mM) (mM) (μg/mL) (ppm) (ppm) (ppm) (ppm) (ppm)control - no enzyme 0.75 1.4 0 1.3 2.7 4.7 7.7 9.7 A. mirum wild-type 40.75 1.4 1.0 2.0 3.6 5.4 6.8 7.5 A. mirum C276S 14 0.75 1.4 1.0 4.6 9.313.0 16.0 17.0 A. mirum C276T 16 0.75 1.4 1.0 5.3 11.1 17.1 21.1 23.1 T.maritima C277S 17 0.75 1.4 1.0 2.9 5.2 8.0 11.1 12.0 T. maritima C277T18 0.75 1.4 1.0 3.4 6.2 9.5 13.2 14.5 A. mirum wild-type 4 0.75 1.4 2.02.1 3.8 5.2 6.3 6.5 A. mirum C276S 14 0.75 1.4 2.0 6.8 12.0 14.9 17.218.7 A. mirum C276T 16 0.75 1.4 2.0 8.6 16.0 20.7 24.4 26.1 T. maritimaC277S 17 0.75 1.4 2.0 3.8 7.0 10.5 13.4 14.4 T. maritima C277T 18 0.751.4 2.0 4.7 8.4 12.8 16.5 17.9

What is claimed is:
 1. A process for producing a peroxycarboxylic acidcomprising: (a) providing a set of reaction components comprising: (1)at least one substrate selected from the group consisting of: (i) one ormore esters having the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=a C1 toC7 linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m=an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁=a C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁=a C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=a C1 toC10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); (2) a source of peroxygen;and (3) an enzyme catalyst comprising a polypeptide having perhydrolyticactivity and an amino acid sequence having at least 80% identity to theamino acid sequence set forth in SEQ ID NO: 8, provided that the aminoacid residue bound to the C-terminal side of the catalytic histidine isnot glutamic acid; (b) combining the set of reaction components undersuitable reaction conditions whereby peroxycarboxylic acid is produced;and (c) optionally diluting the peroxycarboxylic acid produced in step(b).
 2. The process of claim 1 further comprising the step of: d)contacting a hard surface or inanimate object with the peroxycarboxylicacid produced in step (b) or step (c); whereby said hard surface or saidinanimate object is disinfected, bleached, destained or a combinationthereof.
 3. The process of claim 1 wherein the inanimate object is amedical instrument.
 4. The process of claim 1 further comprising thestep of: d) contacting an article of clothing or a textile withperoxycarboxylic acid produced in step (b) or step (c); whereby thearticle of clothing or textile receives a benefit.
 5. The process ofclaim 4 wherein the benefit is selected from the group consisting of adisinfecting, sanitizing, bleaching, destaining, deodorizing, andcombinations thereof.
 6. The process of claim 1 further comprising thestep of: d) contacting wood pulp or paper pulp with peroxycarboxylicacid produced in step (b) or step (c); whereby the wood pulp or paperpulp is bleached.
 7. The process of claim 1 wherein the substrate isselected from the group consisting of: monoacetin; diacetin; triacetin;monopropionin; dipropionin; tripropionin; monobutyrin; dibutyrin;tributyrin; glucose pentaacetate; β-D-galactose pentaacetate, sorbitolhexaacetate, sucrose octaacetate, xylose tetraacetate; acetylated xylan;acetylated xylan fragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,2,5-pentanediol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol,1,6-hexanediol; 4-acetoxybenzoic acid; and mixtures thereof.
 8. Theprocess of claim 7 wherein the substrate is triacetin.
 9. The process ofclaim 1 wherein the peroxycarboxylic acid produced is peracetic acid,perpropionic acid, perbutyric acid, perlactic acid, perglycolic acid,permethoxyacetic acid, per-β-hydroxybutyric acid, or mixtures thereof.10. The process of claim 1 wherein the enzyme catalyst is in the form ofa microbial cell, a permeabilized microbial cell, a microbial cellextract, a partially purified enzyme, or a purified enzyme.
 11. Acomposition comprising: (a) a set of reaction components comprising: (1)at least one substrate selected from the group consisting of: (i) one ormore esters having the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=a C1 toC7 linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m=an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁=a C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁=a C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=a C1 toC10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); (2) a source of peroxygen;and (3) an enzyme catalyst comprising a polypeptide having perhydrolyticactivity and an amino acid sequence having at least 80% identity to theamino acid sequence set forth in SEQ ID NO: 8, provided that the aminoacid residue bound to the C-terminal side of the catalytic histidine isnot glutamic acid; and (b) at least one peroxycarboxylic acid formedupon combining the set of reaction components of (a).
 12. A peracidgeneration and delivery system comprising: (a) a first compartmentcomprising (1) an enzyme catalyst comprising a polypeptide havingperhydrolytic activity and an amino acid sequence having at least 80%identity to the amino acid sequence set forth in SEQ ID NO: 8, providedthat the amino acid residue bound to the C-terminal side of thecatalytic histidine is not glutamic acid; (2) at least one substrateselected from the group consisting of: (i) one or more esters having thestructure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=a C1 toC7 linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m=an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁=a C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁=a C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=a C1 toC10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); and (3) an optional buffer;and (b) a second compartment comprising (1) source of peroxygen; (2) aperoxide stabilizer; and (3) an optional buffer.
 13. The peracidgeneration and delivery system of claim 12 wherein the substratecomprises triacetin.
 14. A laundry care composition comprising a) apolypeptide having perhydrolytic activity and an amino acid sequencehaving at least 80% identity to the amino acid sequence set forth in SEQID NO: 8, provided that the amino acid residue bound to the C-terminalside of the catalytic histidine is not glutamic acid; b) at least onesubstrate selected from the group consisting of: (i) one or more estershaving the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=a C1 toC7 linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m=an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁=a C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁=a C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=a C1 toC10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); and c) a source ofperoxygen; and d) at least one surfactant.
 15. A personal care productcomprising a polypeptide having perhydrolytic activity, said polypeptidehaving an amino acid sequence having at least 80% identity to the aminoacid sequence set forth in SEQ ID NO:8, provided that the amino acidresidue bound to the C-terminal side of the catalytic histidine is notglutamic acid.
 16. The personal care product of claim 15 wherein theproduct is a shampoo, a body lotion, a shower gel, a topicalmoisturizer, a toothpaste, a toothgel, a mouthwash, a mouthrinse, ananti-plaque rinse or a topical cleanser.